Methods and compositions for tissue adhesives

ABSTRACT

Disclosed herein are methods of connecting disrupted tissue, tissue repair, treating colorectal disorder and tissue welding. The methods comprise using a bioadhesive composition comprising ELP and light absorbing chromophores and irradiating the bioadhesive tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit of U.S. Provisional Application No.61/682,129, filed on Aug. 10, 2012, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under HDTRA1-10-1-0109awarded by the Defense Threat Reduction Agency (DTRA). The United Statesgovernment has certain rights in the invention.

BACKGROUND

Surgical procedures that excise tissue or repair of tissues that areseparated or disrupted often require suturing or staples to join thefree ends of the tissue again to form a close seal for healing of thetissues. In some instances, the tissues must be connected tightly enoughto prevent fluids or materials contained on one side of the attachedtissue from leaking through to the other side of the tissue or fromentering a surrounding body cavity. Maintaining the structural integrityof the original tissue at the repair site is a goal in creating therepair site. Surgical repair of gastrointestinal tissues, particularlyafter excision of tissue or growths, often results in repair sites thatdo not sufficiently seal the tissues to prevent leakage of intestinalmaterials into the surrounding peritoneal cavity.

Treatment of colorectal diseases may require surgical interventioninclude such diseases as colorectal cancer and inflammatory boweldisease (IBD) among others. In the United States, approximately 143,000and 1.4 million people suffer from colorectal cancer and inflammatorybowel disease (IBD), respectively (National Cancer Institute and Centersfor Disease Control and Prevention). Thus, every year, over 600,000people in the United States will undergo surgical procedures to treat anumber of colorectal diseases such as colorectal cancer, inflammatorybowel disease (IBD), and diverticulitis (inflammation of pouches formedon the other side of the colon) (^([19, 20)). Patients undergo eitherconventional open surgery or laparoscopic surgeries to remove diseasedtissue^([21]). These patients require end-to-end anastomoses of thehealthy sections following removal of diseased segments (FIG. 1).Surgical suturing and stapling are still the most common and importantprocedures in colorectal anastomoses^([22, 23]). However, both methodsrely on piercing healthy tissue and can cause anastomosis leakage.Leakage of intestinal bacteria from the bacteria-rich colorectal systeminto abdominal lumen can cause serious infection leading to potentiallydeadly peritonitis^([3, 4, 6, 24]). It is reported that one-third of themortality after colorectal surgery is due to anastomosis leakage^([25]).Alternative or supportive anastomosis methods are urgently required inorder to decrease leakage rate and promote tissue regeneration aftercolorectal surgery.

Laser tissue welding (LTW) has emerged as a “sutureless” surgical methodfor the anastomosis of ruptured tissues (e.g. vessels, bowel, urinarytract, skin and others)^([17-15]). In LTW, laser light is absorbed bythe tissue, which converts it into heat energy, resulting in thealteration of tissue proteins^([14, 16]). Fusing of the photothermallyaltered tissue proteins via covalent and electrostaticinteractions^([11, 17, 18]) is thought to be the primary mechanismresponsible for welding (fusing) tissues. However, the efficacy of LTWis severely restricted due to lack of effective bioadhesives that fusetissues. Specifically, laser irradiation of tissues can result inphotothermal conversion of light to heat, resulting indenaturation/structural change of proteins, which fuse at the weld site.This process results in improved tensile strength of the closure andminimized peripheral tissue destruction. The advantages oflaser-assisted tissue welding (LTW) over conventional suturing andstapling include short operation times, immediate fluid-tight sealing,reduced foreign-body reactions (e.g. inflammatory response) and scarformation and accelerated healing^([10, 26-30]). Concerns associatedwith LTW include insufficient anastomoses strengths due to sub-optimalbioadhesive performance, limited light penetration depth, and peripheraltissue thermal damage.

Accordingly, there is a need for materials and methods that can connectdisrupted tissue using a light source. Such materials and methods aredisclosed herein.

SUMMARY

In accordance with the purpose(s), as embodied and broadly describedherein, in one aspect, relates to connection of tissue, welding tissue,tissue repair and treating colorectal disorders.

Disclosed herein is a method of connecting disrupted tissue, comprising,a) applying an effective amount of a photothermally responsivebioadhesive composition comprising an ELP and a light absorbingchromophore to disrupted tissue in need of being connected; and b)applying an effective amount of a directed light beam to thephotothermally responsive bioadhesive composition and/or the tissue.

In one aspect, instead of using light absorbing chromophore, in themethods described herein, energy absorbing materials, such as magneticparticles can be used. The particles can be nanoparticles. Suitablemagnetic particles include, but are not limited to iron nanoparticles(iron oxide). The magnetic particles can be magnetothermal particles.When such particles are used directed magnetic or radio frequencymethods are used rather than a directed light beam as described in themethods herein.

Also disclosed herein is a method of laser tissue welding, comprising,a) applying an effective amount of a photothermally responsivebioadhesive composition comprising an ELP and a light absorbingchromophore to disrupted tissue in need of being welded; and b) applyingan effective amount of a directed light beam to the photothermallyresponsive bioadhesive composition and/or the tissue.

Also disclosed herein is a method of tissue repair, comprising, a)suturing a tissue with fibers comprising a photothermally responsivecomposition comprising an ELP and a light absorbing chromophore; and b)optionally, applying an effective amount of a photothermally responsivebioadhesive composition comprising an ELP and a light absorbingchromophore to the sutured site; and c) applying an effective amount ofa directed light beam to the sutures and/or to the tissue, andoptionally to the photothermally responsive bioadhesive composition.

Also disclosed herein is a method of treating colorectal disease,comprising, a) applying an effective amount of a photothermallyresponsive bioadhesive composition comprising an ELP and a lightabsorbing chromophore to disrupted colorectal tissue; and b) applying aneffective amount of a directed light beam to the photothermallyresponsive bioadhesive composition and/or the tissue.

In one aspect, the bioadhesive composition can comprise at least 0.5%,1%, 2%, 4%, 5% or 8% of a light absorbing chromophore. The preferredloading is approximately 5% of light absorbing chromophores. Thechromophores may be physically entrapped within the ELP or chemicallyconjugated to the ELP.

In one aspect, the heat generated from the light absorbing chromophoreproduces a bulk temperature, such as a tissue temperature and/orbioadhesive temperature, of at least 55° C., 60° C., 65° C., 70° C., 75°C., 80° C., 85° C. or 90° C.

In one aspect, the light absorbing chromophore crosslink the ELP throughthe heat generated from the absorption of energy through the directedlight beam. In one aspect, the crosslinking connects disrupted tissueand can help the healing of wounds or cuts.

In one aspect, the light absorbing chromophore can comprise silvernanoparticles, gold nanorods, or gold nanoparticles, or mixturesthereof.

In one aspect, an ELP comprises cysteine residues. An ELP can, forexample, comprise at least 2, 4, 6, 8, 10, 12 or 14 cysteine residues.In one aspect, the ELP can comprise at least 8 or 12 cysteine residues.In one aspect, the ELP comprises 8 or 12 cysteine residues in thesequence: MVSACRGPG-[VG VPGVG VPGVG VPGVG VPGVG VPG]₈-[VG VPGVG VPGVGVPGCG VPGVG VPG]₈-WP (SEQ ID NO:1) or MVSACRGPG-[VG VPGVG VPGVG VPGVGVPGVG VPG]₈-[VG VPGVG VPGVG VPGCG VPGVG VPG]₁₂-WP (SEQ ID NO:2).

In one aspect, the bioadhesive composition can reproducibly producetissue temperature of at least 65° C. upon irradiation from a lightsource.

In one aspect, the bioadhesive composition can have anti-microbialproperties, such as anti-bacterial properties. For example, thebioadhesive composition can comprise an anti-bacterial agent. Suitableantibacterial agents include, but are not limited to, MMP inhibitors,small-molecule drugs, peptides, and silver nanoparticles.

In one aspect, the directed light beam can be a laser, such as aTitanium-Sapphire laser, Krypton laser, Ruby laser, Chromium dopedchrysoberyl (alexandrite) laser, Divalent samarium doped calciumfluoride (Sm:CaF₂) laser, AlGaInP laser, AlGaAs laser, Vertical cavitysurface emitting laser (VCSEL). In one aspect, the tissue in need ofbeing welded is from a cut or soar. In another aspect, the in need ofbeing welded is from surgery.

In one aspect, the bioadhesive composition is suitable for suturing. Forexample, the bioadhesive tissue can be in the form a sting or othersuitable for suturing. For example, the bioadhesive composition is inthe form of a fiber.

In one aspect, the directed light beam is a laser, such as aTitanium-Sapphire laser. In one aspect, the wavelength of the light fromthe directed light source is in the near infrared region of the lightabsorption spectrum.

In another aspect, the bioadhesive composition further comprises cells.Suitable cells include but are not limited to, NCM460, fibroblasts, stemcells or mixtures thereof.

In one aspect, the disrupted tissue in need of being welded is from soreor cut. For example, the disrupted tissue in need of being welded isfrom a surgical cut.

In one aspect, the disrupted colorectal tissue is from a section of thecolon that has been removed during surgery.

While aspects of the present invention can be described and claimed in aparticular statutory class, such as the system statutory class, this isfor convenience only and one of skill in the art will understand thateach aspect of the present invention can be described and claimed in anystatutory class. Unless otherwise expressly stated, it is in no wayintended that any method or aspect set forth herein be construed asrequiring that its steps be performed in a specific order. Accordingly,where a method claim does not specifically state in the claims ordescriptions that the steps are to be limited to a specific order, it isno way intended that an order be inferred, in any respect. This holdsfor any possible non-express basis for interpretation, including mattersof logic with respect to arrangement of steps or operational flow, plainmeaning derived from grammatical organization or punctuation, or thenumber or type of aspects described in the specification.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several aspects and together withthe description serve to explain the principles of the invention.

FIGS. 1A-1C show a schematic of laser tissue welding (LTW) usingPlasmonic Nanoparticle (PNP)-ELP nanocomposites for end-to-endcolorectal anastomosis after surgical resection. (A) Removal of diseasedcolon. (B) LTW or combined suturing and LTW using acellular orcellularized PNP-ELP nanocomposite. (C) Fluid-tight sealing with reducedleakage and accelerated healing.

FIG. 2 shows a schematic of GNR-ELP nanocomposite formation. (A)liquid-phase GNR dispersion (B) C₁₂ELPs (ELPs with 12 cysteines withinthe polypeptide sequence) were self-assembled on gold nano rods (GNRs)resulting in GNR-ELP nanoassemblies. (C) Increasing the dispersiontemperature of nanoassemblies above C₁₂ELP T_(t) resulted in theformation of ELP-GNR aggregates (coacervation). (D, E) Coalescence ofthe aggregates and precipitation resulted in nanocomposite formation asshown in E. Environmental field-emission scanning electron microscopyconfirmed uniform GNR distribution within the nanocomposite.

FIGS. 3A-3E show nanocomposite characterization. (A) Digital image, (B)phase contrast image (C) dark-field mage of GNR-ELP nanocomposites. (D)Photothermal response of nanocomposite upon laser exposure; temperaturesof up to 43° C. were reached in −10 min. The nanocomposites containedNIR-absorbing GNRs (E) Diffusion (left) and laser-triggered (right)release of drug (17AAG) from the nanocomposite.

FIGS. 4A-4C show the mechanical properties of nanocomposites at variousGNR weight ratios. (A) Swelling ratio. (B) Absolute shear modulus. (C)Loss angle.

FIGS. 5A-5C show the light absorption spectra of GNR (30 μg)-ELP (1.5mg) (maroon (shown as dark in figure) inset; left), silver nanoparticleor AgNP (15 μg-ELP (2 mg) (silvery white (shown as bright infigure)inset; middle), and hybrid GNR (30 μg)-AgNP (15 μg-ELP (2 mg)(light red inset (shown as dark in figure); right) nanocomposites.Nanocomposites retain the plasmonic properties of the nanoparticles.GNRs (˜15 nm diameter, ˜50 nm length) and spherical AgNP (˜60 nmradius).

FIGS. 6A-6C show the determination of breaking force afternanocomposite-assisted laser tissue welding of porcine intestine. (A).Full thickness incision (˜8 mm in width) was applied at the center ofthe rectangular intestine sections (˜0.1 cm thick, 4×1 cm). The GNR-ELPnanocomposite was applied on top of the serosa layer and across the fullthickness incision with full contact. Laser light (20 W/cm², 1mm/second) was applied vertically. (B) The maximum force (N) achievedprior to sample breaking was recorded and reported as ultimate tensilestrength (UTS, kPa). n=1-12. (C) Ultimate tensile strength of tissuesbefore and after laser tissue welding using cellularized nanocomposites.Fibroblasts were cultured on top of nanocomposites for 1, 4 and 7 days.

FIGS. 7A-7C show GNR-ELP nanocomposite cellularized with 3T3 murinefibroblasts. Fluorescence (Live-Dead stain) and confocal microscopy showthe viability of encapsulated cells at 72 h (live cells: brighter).

FIGS. 8A-8D show (A) Schematic of cellularized PNP-ELPnanocomposite-mediated LTW after colorectal surgery. Viable cellsoutside the laser path can promote regeneration after anastomosis. (B)PC3-PSMA cells in the laser path (dotted line) on GNRELP nanocompositedie (red) due to hyperthermia; peripheral cells are alive (green). (C)Cell proliferation (mm) from nanocomposite. C1 and C2 show cellmigration on day 2 and day 3 (phase contrast microscopy). (D)Proliferation distance in mm versus time in days is shown in C3.

FIGS. 9A-9H show the formation of C8 elastin-like polypeptide-goldnanorod and C₁₂ elastin-like polypeptide-gold nanorod matrices.C₈ELP-GNR, C_(t2)ELP-GNR, C₈ELP alone and GNR alone were incubated in awater bath (37° C.) for 6 h, followed by cooling and storage at roomtemperature. The transition temperature of C₈ELP was 31.3° C. and thatof C₁₂ELP was 30.5° C. Digital snapshots of C₈ELP-GNR, and C₁₂ELP-GNRformation were taken at (A) 0 min, (B) 15 min (0.25 h), (C) 6 h and (D)24 h. Images (E) and (F) show the C₈ELP-GNR and C₁₂ELP-GNR matrices. Twocontrols, C₈ELP alone and GNR alone, are shown in (G) 0 min and (H) 6 h;no matrices were formed in these cases; as expected, an increase inC₈ELP turbidity was observed following an increase in temperature (H).ELP: Elastin-like polypeptide; GNR: gold nanorod

FIG. 10 shows the formation and dissolution kinetics of C₈ elastin-likepolypeptide-gold nanorod and C₁₂ elastin-like polypeptide-gold nanorodmatrices. The UV-vis absorbance spectra of the supernatant of CsELP-GNRand C₁₂ELP-GNR were monitored during the 37° C. water bath incubationstage (0-6 h), cooling/storage (6-24 h), and 30 min after addingdithiothreitol solution (24.5 h). Increase in optical density of GNRspectrum in the first hour indicates turbidity due to ELP aggregationabove the transition temperature. The flat absorbance spectrum at 6 hindicates completion of the matrix formation and the absence of GNRs inthe supernatant. Full recovery of the GNR spectrum at 24.5 h indicatesdegradability of the matrix in the presence of reducing agents.

FIG. 11 shows C₈ elastin-like polypeptide-gold nanorod and C₁₂elastin-like polypeptide-gold nanorod matrices in the presence andabsence of dithiothreitol (DTT). C₈ELP-GNR, C₁₂ELP-GNR matrices wereformed by incubation at 37° C. for 6 h as described above, followed bycooling and storage at room temperature (6-24 h). The supernatant wasreplaced with an equal volume of 10 mM DTT for 30 min at 4° C. (24-24.5h). (A & B) C₈ELP-GNR and C₁₂ELP-GNR matrices with (A) and without (B)the presence of DTT.

FIG. 12 shows the Fourier-transform infrared (FT-IR) spectrum of goldnanorod, C₈ elastin-like polypeptide-gold nanorod and C₁₂ elastin-likepolypeptide-gold nanorod matrices. Fourier-transform infrared (BrukerVacuum FT-IR IFS 66v/S) spectroscopy of CsELP-GNR and C₁₂ELP-GNRmatrices indicating characteristic peaks at wave numbers of 3500-3000cm¹, corresponding to the N—H stretching vibrations, 1660 cm¹,corresponding to the C═O stretches in the amide functionality (amide Ipeak), and a peak at 1550 cm¹, which is the combination band of N—Hbending and C—N stretching vibrations (amide II peak).Cetyltrimethylammonium bromide(CTAB)-GNR shows peaks at 2850 and 2917cm¹, which reflect the symmetric and asymmetric C—H stretchingvibrations, respectively. No peaks were observed in the 2550-2600 cm¹region, which indicates the absence of the S—H bond

FIG. 13 shows scanning electron microscope images of C₈ elastin-likepolypeptide-gold nanorod and C₁₂ elastin-like polypeptide-gold nanorodmatrices. Environmental field-emission scanning electron microscopy(Philips FEI XL-30 scanning electron microscope operating anaccelerating voltage of 25 kV) images indicate a fairly uniformdistribution of GNRs throughout C₈ELP-GNR and C₁₂ELP-GNR matrices.

FIG. 14 shows optical and photothermal response of C12 elastin-likepolypeptide-gold nanorod matrices. C12ELP-GNR matrices were formed on aglass cover slip (inset) and the absorbance (optical density) spectrumof the film was analyzed using a plate reader. Uniform distribution ofGNRs in the matrix resulted in optical properties similar to that of GNRdispersions; characteristic peaks at 520 nm and in the near infrared(˜850 nm) region in the spectrum can be seen. Retention of opticalproperties of GNRs in the matrix resulted in a reliable photothermalresponse as seen from the temperature kinetics of 500 ulphosphate-buffered saline on top of the matrix in a 24-well plate. Thematrix was irradiated using an 850 nm laser at two different powerdensities and the temperature of phosphate-buffered saline was measuredusing a K-type thermocouple.

FIG. 15 shows spectrum of 17-AAG-loaded C₁₂ elastin-likepolypeptide-gold nanorod matrices. C₁₂ELP-GNR matrices loaded with theheat-shock protein (HSP) 90 inhibitor drug, 17-AAG for ablation ofcancer cells using a combination treatment of hyperthermia and HSPinhibition. (A) Absorbance spectrum 17-AAG shows a peak at approximately330 nm, which is reflected in the 17-AAG-C₁₂ELP-GNR matrices. (B) Theinset in (B) shows a digital snapshot of the 17-AAG-C₁₂ELP-GNR matrixloaded with 0.55 mg of the HSP90 inhibitor drug. Drug loading can alsobe seen from the change in color of the matrix.

FIG. 16 shows photothermally activated release of the heat-shock protein90 inhibitor drug, 17-AAG from 17-AAG-C₁₂ elastin-like polypeptide-goldnanorod matrices. (A) Diffusional release (leaching) of 17-AAG from the17-AAG-C₁₂ELP-GNR matrix was monitored for 24 h to a supernatant ofphosphate-buffered saline (volume 500 μl). Approximately 10 μg of thedrug was released in the first hour, following which minimal drugrelease was observed for the rest of the duration as seen in thecumulative data plotted above. The matrix was then subjected to laserirradiation for releasing the drug. During laser pulse-triggeredrelease, the first laser irradiation lasted for 5 min (850 nm, 25W/cm²), the second to sixth laser irradiations (850 nm, 25 W/cm²) werefor 10 min, with 20-min interval without laser in between eachirradiation. Data shown are mean±standard deviation from fourindependent experiments (n=4). (B) The temperature profile during 10-minlaser exposure was monitored with a K-type thermocouple. The temperaturereached 43-44° C. (heat-shock conditions) following 5 min of laserirradiation, and remained invariant thereafter. Laser irradiationresulted in an additional 45-50 mg of 17-AAG released from the17-AAG-C₁₂ELP-GNR matrices, indicating the potential for combinedhyperthermia and heat-shock inhibition. (C) Role of laser irradiation onenhancing 17-AAG release from C₁₂ELP-GNR matrices. Matrices were firstinvestigated for diffusional leaching of the drug, followed byincubation in a water bath at 42° C., and were finally irradiated withan near-infrared laser. Increased amounts of drug were releasedfollowing laser treatment, presumably due to higher localizedtemperatures in the path of the laser. 17-AAG:17-(allylamino)-17-demethoxygeldanamycin.

FIG. 17 shows C12 elastin-like polypeptide-gold nanorod matrices cellablation set up with fluorescence (A & B) and phase (C & D) images.PC3-PSMA human prostate cancer cells were cultured on C12ELP-GNRmatrices (without 17-AAG drug) for 24 h, and irradiated with an 850-nmlaser (25 W/cm2) for 7 min. Viability of cells directly under the 1-mmlaser irradiation spot (top; circled area) and outside the laser spot(bottom) was determined using the Live/Dead® assay in which living cellsstained green while dead cells stained red. Cells directly under thelaser were killed by the hyperthermic treatment while those outside thelaser irradiation spot were alive. Approximate locations of the imageson the matrix are shown. Representative images are from two independentexperiments (n=2). Scale bar: 500 μm.

FIG. 18 shows 17-AAG-C12 elastin-like polypeptide-gold nanorod matricescell culture set up with fluorescence (A & B) and phase (C & D) images.PC3-PSMA human prostate cancer cells were cultured on 17-AAG-C₁₂ELP-GNRmatrices for 48 h (no laser treatment) and viability of cells wasdetermined using the Live/Dead® assay. Diffusional release of 17-AAGfrom the matrix did not alter the viability of the cells. Approximatelocations of the images on the matrix are shown. Representative imagesfrom three independent experiments (n=3). Scale bar: 500 μm.

FIG. 19 shows 17-AAG-C₁₂ elastin-like polypeptide-gold nanorod matricescell ablation set up with fluorescence (A & B) and phase (C & D) images.PC3-PSMA human prostate cancer cells were cultured on C₁₂ELP-GNR-17-AAGmatrices for 24 h and irradiated with an 850-nm laser (25 W/cm²) for 7min. Cell viability was investigated after 24 h of the laser treatmentusing the Live/Dead® assay (total cell culture time=48 h).Representative images at two different locations away from the laserirradiation spot demonstrate approximately 90% cell death due to thecombination of mild hyperthermia (43° C.; FIG. 16 at (B)) and release ofthe heat-shock inhibitor drug 17-AAG (FIG. 16 at (A)). This pattern ofuniform cell death throughout the well is higher than what was seen withthe single agent (i.e., mild hyperthermia alone and 17-AAG releasealone) treatments. Approximate locations of the images are on the matrixare shown. Representative images from three independent experiments(n=3). Scale bar: 500 μm.

FIG. 20 shows the quantitative analysis of cell death demonstrates theefficacy of the laser-induced combination treatment of hyperthermia andheat-shock inhibitor 17-AAG using 17-AAG-C12 elastin-likepolypeptide-gold nanorod matrices. Matrix indicates the C₁₂ELP-GNRmatrix and 17-AAG matrix indicates 17-AAG-C₁₂ELP-GNR (i.e., drug-loaded)matrices (n=3 for all conditions).

FIG. 21 shows the bursting and leakage pressures of tissues before andafter laser tissue welding using nanocomposite solders.

FIGS. 22A-22B show (A) and (B) the change in OD₆₀₀ of fresh LB brothmonitored as a function of time (A: 0-8 h, B: 0-24 h) and at differenttreatment conditions for quantitative comparison.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or can be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

DESCRIPTION

The present invention can be understood more readily by reference to thefollowing detailed description of the invention and the Examplesincluded therein.

Before the present compounds, compositions, articles, systems, devices,and/or methods are disclosed and described, it is to be understood thatthey are not limited to specific synthetic methods unless otherwisespecified, or to particular reagents unless otherwise specified, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only andis not intended to be limiting. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, example methods andmaterials are now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedherein can be different from the actual publication dates, which canrequire independent confirmation.

1. Definitions

As used herein, nomenclature for compounds, including organic compounds,can be given using common names, IUPAC, IUBMB, or CAS recommendationsfor nomenclature. When one or more stereochemical features are present,Cahn-Ingold-Prelog rules for stereochemistry can be employed todesignate stereochemical priority, E/Z specification, and the like. Oneof skill in the art can readily ascertain the structure of a compound ifgiven a name, either by systemic reduction of the compound structureusing naming conventions, or by commercially available software, such asCHEMDRAW™ (Cambridgesoft Corporation, U.S.A.).

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a functionalgroup,” “an alkyl,” or “a residue” includes mixtures of two or more suchfunctional groups, alkyls, or residues, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, a further aspect includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms a further aspect. It willbe further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that each unit between two particularunits are also disclosed. For example, if 10 and 15 are disclosed, then11, 12, 13, and 14 are also disclosed.

References in the specification and concluding claims to parts by weightof a particular element or component in a composition denotes the weightrelationship between the element or component and any other elements orcomponents in the composition or article for which a part by weight isexpressed. Thus, in a compound containing 2 parts by weight of componentX and 5 parts by weight component Y, X and Y are present at a weightratio of 2:5, and are present in such ratio regardless of whetheradditional components are contained in the compound.

A weight percent (wt. %) of a component, unless specifically stated tothe contrary, is based on the total weight of the formulation orcomposition in which the component is included.

As used herein, the term “bulk temperature” or the like terms refer tothe temperature that is produced in a material, i.e., bioadhesivecomposition, from the heat generated from a light absorbing chromophoreonce irradiated with light. For example, a “bulk temperature” can be thetemperature in a bioadhesive composition that was generated from goldnanorods upon exposure to a laser. The temperature of the bioadhesivecomposition or a portion thereof can have a bulk temperature. Also thetemperature in the tissue surrounding the bioadhesive composition canhave a bulk temperature.

As used herein, the terms “optional” or “optionally” means that thesubsequently described event or circumstance can or cannot occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, the term “photothermally responsive bioadhesivecomposition” or the like terms refer to a bioadhesive composition thatcan be heated once irradiated with a directed light beam. For example, acomposition of ELP and a light absorbing chromophore that heats up onceirradiated with a laser is a photothermally responsive bioadhesivecomposition.

As used herein, the term “encapsulated in” or the like terms refer towhen individual particles/cells are incorporated within a network, sucha as a ELP network, which forms the nanocomposite. This can beaccomplished by mixing cells with soluble polypeptide (liquid) and theninducing a phase change from the liquid phase to solid phase in order tophysically entrap cells within the interconnected ELP matrix

Unless otherwise expressly stated, it is in no way intended that anymethod set forth herein be construed as requiring that its steps beperformed in a specific order. Accordingly, where a method claim doesnot actually recite an order to be followed by its steps or it is nototherwise specifically stated in the claims or descriptions that thesteps are to be limited to a specific order, it is no way intended thatan order be inferred, in any respect. This holds for any possiblenon-express basis for interpretation, including: matters of logic withrespect to arrangement of steps or operational flow; plain meaningderived from grammatical organization or punctuation; and the number ortype of embodiments described in the specification.

Disclosed are the components to be used to prepare the compositions ofthe invention as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds can not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular compound is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the compounds are discussed, specifically contemplated is eachand every combination and permutation of the compound and themodifications that are possible unless specifically indicated to thecontrary. Thus, if a class of molecules A, B, and C are disclosed aswell as a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited each is individually and collectively contemplated meaningcombinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considereddisclosed. Likewise, any subset or combination of these is alsodisclosed. Thus, for example, the sub-group of A-E, B-F, and C-E wouldbe considered disclosed. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the compositions of the invention. Thus, if there are avariety of additional steps that can be performed it is understood thateach of these additional steps can be performed with any specificembodiment or combination of embodiments of the methods of theinvention.

It is understood that the compositions disclosed herein have certainfunctions. Disclosed herein are certain structural requirements forperforming the disclosed functions, and it is understood that there area variety of structures that can perform the same function that arerelated to the disclosed structures, and that these structures willtypically achieve the same result.

2. Methods

The present invention comprises methods and compositions for repair oftissues and organs, such as tissues or organs that have undergonedisruption of the tissue or organ such as tears, removal of sections ofthe tissue or organ, or addition of cellular or acellular materials tothe tissue or organ. Disclosed herein are methods for repair ofdisrupted gastrointestinal organs, such as the colon, large bowel, orsmall bowel, but the present invention is not limited to only thedisclosed applications of the methods, but it is contemplated othertissues and organs could benefit from the same repair methods. As usedherein, a “disrupted” tissue or organ is a tissue or organ that hasundergone a tear or cut, whether by intention, such as in surgery, or byaccident, such as in a traumatic event, such that free ends or freesurfaces are created in what was once a whole tissue or organ. Forexample, two free ends in the colon are created when a section of thecolon is removed surgically. In general surgical methods, the two freeends may be joined to each other with sutures to form a repair site.Methods disclosed herein may be used to join the two free ends to eachother to form a repair site. In some instances, one free end may becreated by a surgical or accidental event, and the one free end may beclosed by methods disclosed herein.

Laser-assisted colorectal anastomoses can provide immediate fluid-tightsealing upon treatment, and may reduce the frequency of colonicanastomosis leakage^([35]). Cilesiz et al. reported that Ho:YAG andargon laser welding of rat intestine resulted in a comparable burstingpressure and healing rate to suture anastomoses^([36, 37]). In a caninejejunum study, strong tissue fusion was not possible at or below asurface temperature of 70° C., but was accomplished above 80° C.^([26]).Lauto et al. reported genipin-crosslinked albumin can significantlyincrease the tensile strength of adhesive-tissue bonds after laserwelding^([35]). TGF-β, a key component in the fibrogenic process andinflammatory response, was incorporated into human albumin solder toaccelerate wound healing process after laser welding^([38]). Overall, itis recognized that temperatures above 60° C. are necessary to providerobust closure^([39]). The ability to precisely deliver laser energy isimportant in LTW^([26, 39-41]). Additionally, matrix metalloproteinase(MMP) over expression is common and causes tissue degradation duringearly stages of colon repair^([42, 43]).

The light dosage required to induce similar thermal response using goldnanostructures is 10- to 25-fold lower than with photoabsorbingdyes^([45]). GNRs, possess among the highest near infrared (NIR)absorption efficiencies^([46]) and can convert light into heat mostefficiently compared to other gold nanoparticles. Moreover, light in NIRregion demonstrates maximal tissue penetration, due to minimal lightabsorption by water and bloody^([47]). Silver nanoparticles are alsoexcellent photothermal convertors.

Although PNP-ELP based LTW may occur on exposed tissue during surgery,administration of NIR light is possible in the colon in vivo^([48])including using endoscopes or catheters^([49]). NIR light enables deeperpenetration and localization of heat in the tissue. Further, use ofplasmonic nanoparticles can mean high localization of heat energyleading to greater welding efficacies, and reduction in unwantedperipheral thermal damage^([33, 34]). Silver (Ag), including Agnanoparticles (AgNP)^([67-69]), demonstrates antibacterialproperties^([70]).

ELPs are reported to be biocompatible with low immunogenicities^([50])and have been explored for diverse applications^([51-53]), includingtreatment of chronic wounds in vivo^([54]). However, low dynamic shearstiffness associated with ELP coacervates can limit their application inregenerative medicine, specifically, in cases where significant loadsupport may be required^([55]). Crosslinking ELPs with metallicnanoparticles can provide improved dynamic shear stiffness as well asstretch/recoil properties with minimal immunogenicity, all of which aresignificant in colon anastamoses.

Disclosed herein is a method of connecting disrupted tissue, comprising,a) applying an effective amount of a photothermally responsivebioadhesive composition comprising an ELP and a light absorbingchromophore to disrupted tissue in need of being connected; and b)applying an effective amount of a directed light beam to thephotothermally responsive bioadhesive composition and/or the tissue.

Also disclosed herein is a method of laser tissue welding, comprising,a) applying an effective amount of a photothermally responsivebioadhesive composition comprising an ELP and a light absorbingchromophore to disrupted tissue in need of being welded; and b) applyingan effective amount of a directed light beam to the photothermallyresponsive bioadhesive composition and/or the tissue.

Also disclosed herein is a method of tissue repair, comprising, a)suturing a tissue with fibers comprising a photothermally responsivecomposition comprising an ELP and a light absorbing chromophore; and b)optionally, applying an effective amount of a photothermally responsivebioadhesive composition comprising an ELP and a light absorbingchromophore to the sutured site; and c) applying an effective amount ofa directed light beam to the sutures and/or to the tissue, andoptionally to the photothermally responsive bioadhesive composition.

Also disclosed herein is a method of treating colorectal disease,comprising, a) applying an effective amount of a photothermallyresponsive bioadhesive composition comprising an ELP and a lightabsorbing chromophore to disrupted colorectal tissue; and b) applying aneffective amount of a directed light beam to the photothermallyresponsive bioadhesive composition and/or the tissue.

In one aspect, instead of using light absorbing chromophore, in themethods described herein, energy absorbing materials, such as magneticparticles can be used. The particles can be nanoparticles. Suitablemagnetic particles include, but are not limited to iron nanoparticles(iron oxide). The magnetic particles can be magnetothermal particles.When such particles are used directed magnetic or radio frequencymethods are used rather than a directed light beam as described in themethods herein.

In one aspect, the bioadhesive composition can comprise at least 0.5%,1%1 3%, 5%, 10%, 15%, 20%, 25% or 30% of a light absorbing chromophore.For example, the bioadhesive composition can comprise at least 0.5%, 1%,2%, 4%, 5% or 8% of a light absorbing chromophore. The preferred loadingwas suggested at approximately 5% of light absorbing chromophores.

In another aspect, the bioadhesive composition can comprise betweenabout 0.5%, 1%, 2%, 4%, 5%, or 8% of a light absorbing chromophore. Forexample, the bioadhesive composition can comprise between about 2%-8%and 3%-6% of a light absorbing chromophore.

In one aspect, the light absorbing chromophore generates heat once itabsorbs light of an appropriate wavelength. For example, gold nanorodsgenerate heat once they absorb light from a laser, such as a argonlaser. In one aspect, the heat generated from the light absorbingchromophore produces a bulk temperature, such as a tissue temperatureand/or bioadhesive temperature, of at least 55° C., 60° C., 65° C., 70°C., 75° C., 80° C., 85° C. or 90° C. Preferably, the heat generated fromthe light absorbing chromophore produces a tissue temperature orbioadhesive temperature of at least 65° C. In one aspect, the bulktemperature can be reproducibly produced.

In one aspect, the light absorbing chromophores crosslink the ELPthrough the heat generated from the absorption of energy through thedirected light beam. In one aspect, the crosslinking connects disruptedtissue and can help the healing of wounds or cuts.

In one aspect, the light absorbing chromophore can comprise silvernanoparticles, gold nanorods, or gold nanoparticles, or mixturesthereof. For example, the light absorbing chromophore can comprise goldnanorods. In another example, the light absorbing chromophore cancomprise gold nanorods, gold nanospheres, gold nanoshells, goldnanocubes, and silver nanoparticles.

In one aspect, the light absorbing chromophore has anti-microbialproperties, such as anti-bacterial properties. For example, the lightabsorbing chromophore can be silver nanoparticles. The light absorbingchromophore can have effective antibacterial activities, for example,against E. coli and Staphylococcus aureus, shown by using the agarKirby-Bauer disk-diffusion method.

In one aspect, an ELP comprises cysteine residues. An ELP can, forexample, comprise at least 2, 4, 6, 8, 10, 12 or 14 cysteine residues.Thus, an ELP can, for example, comprise at least 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12 cysteine residues. In one aspect, an ELP can, for example,comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cysteine residues. Forexample, the ELP can comprise at least 8 or 12 cysteine residues. An ELPhaving 8 cysteine residues can be referred to as C₈ELP. An ELP having 12cysteine residues can be referred to as C₁₂ ELP. In one aspect, the ELPcomprises 8 or 12 cysteine residues in the sequence: MVSACRGPG-[VG VPGVGVPGVG VPGVG VPGVG VPG]₈-[VG VPGVG VPGVG VPGCG VPGVG VPG]₈-WP (SEQ IDNO:1) or MVSACRGPG-[VG VPGVG VPGVG VPGVG VPGVG VPG]₈-[VG VPGVG VPGVGVPGCG VPGVG VPG]₁₂-WP (SEQ ID NO:2).

For example, cetyltrimethyl ammonium bromide (CTAB) surfactant-templatedgold nanorods (GNRs) can be used to facilitate the irreversiblecrosslinking of cysteine-containing ELPs leading to the formation ofELP-GNR nanocomposites. These ELP-GNR nanocomposites not only retainedthe photothermal properties of gold nanorods, are also able to act as‘depots’ for drug release.

In one aspect, the bioadhesive composition, such as a PNP-ELPnanocomposite, can reproducibly produce tissue temperature of at least55° C. upon irradiation from a light source, for example a laser. Forexample, the bioadhesive composition can reproducibly produce tissuetemperature of at least 55° C., 60° C., 65° C., 70° C., 75° C., 80° C.,85° C. or 90° C. upon irradiation from a light source, for example alaser. In one aspect, the bioadhesive composition can reproduciblyproduce tissue temperature of at least 65° C. upon irradiation from alight source.

In one aspect the bioadhesive composition can have anti-microbialproperties, such as anti-bacterial properties. For example, abioadhesive composition can comprise an anti-bacterial agent. Suitableantibacterial agents include, but are not limited to, amikacin,gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin,geldanamycin, herbimycin, loracarbef, ertapenem, doripenem, imipenem,cilastatin, meropenem, cefadroxil, cefazolin, cefalotin, cefalexin,cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime,cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime,ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftarolinefosamil, ceftobiprole, teicoplanin, vancomycin, telavancin, clindamycin,lincomycin, daptomycin, azithromycin, clarithromycin, dirithromycin,erythromycin, roxithromycin, troleandomycin, telithromycin,spectinomycin, spiramycin, aztreonam, furazolidone, nitrofurantoin,azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin,mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillinV, piperacillin, penicillin G, temocillin, ticarcillin, amoxicillin,ampicillin, piperacillin, ticarcillin, bacitracin, colistin, polymyxinB, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin,moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin,grepafloxacin, sparfloxacin, temafloxacin, mafenide, sulfacetamide,sulfadiazine, silver sulfadiazine, sulfamethizole, sulfamethoxazole,sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim,trimethoprim-sulfamethoxazole (co-trimoxazole) (TMP-SMX),demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline,clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide,isoniazid, pyrazinamide, rifampicin rifabutin, rifapentine,streptomycin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid,linezolid, mtronidazole, mupirocin, platensimycin, quinupristin,dalfopristin, rifaximin, thiamphenicol, tigecycline, tinidazole, andanti-microbial peptides and proteins.

In one aspect the bioadhesive composition may comprise active agents.For example, an active agent may be an MMP inhibitor. For example, anMMP inhibitor can be doxycycline. MMP inhibitors include tissueinhibitor of metalloproteinases (TIMPs), TIMP-1, TIMP-2, TIMP-3, andTIMP-4; zinc chelating groups including hydroxamates, carboxylates,thiols, and phosphinyls, Minocycline, marimastat (BB-2516), abroad-spectrum MMP inhibitor, and cipemastat (Ro 32-3555), an MMP-1selective inhibitor.

MMP overexpression breaks down early tissue formed during anastomoses inthe colon, which compromises healing^([42, 56]). Thus, the bioadhesivecompositions disclosed herein can be employed for diffusion-based aswell as laser-triggered localized delivery of MMP inhibitors (e.g.doxycycline). Other suitable active agents include, but are not limitedto a soluble factors, such as cytokines or growth factors. For example,a soluble factor can comprises FGF (fibroblast growth factor), TGF-beta,EGF, VEGE, or other factors known as growth factors or cytokines, orknown to be involved in wound healing and repair. In another aspect, theactive agent can be encapsulated. In another example, the active agentis not encapsulated. In one aspect, the active agent is located withinthe bioadhesive composition.

In one aspect, the bioadhesive composition can comprise extracellularmatrix material(s). Non-limiting examples of extracellular matrixmaterial(s) include collagen and/or silk proteins. Thus, the bioadhesivecomposition can comprise collagen and/or silk proteins.

In one aspect, the bioadhesive composition can comprise ELP-collagen,ELP-silk, ELP-fibrin, or ELP-polymer, and conjugates and blends thereof.For example, the bioadhesive composition can comprise ELP-collagen. Inanother example, the bioadhesive composition can comprise ELP-silk. Inyet another example, the bioadhesive composition can compriseELP-fibrin. In yet another example, the bioadhesive composition cancomprise ELP-polymer.

In one aspect, the directed light beam can be a laser, such as an argonlaser. The directed light beam preferably has a wavelength that iscompatible with the light absorbing chromophore. The light absorbingchromophore absorbs the energy from the directed light beam and reliablyheats up to desired temperature. The intensity of the direct light beamcan be adjust to achieve suitable temperatures in the tissue andbioadhesive material, such as 65° C., 70° C., 75° C., 80° C., 85° C., or90° C. It is possible to tailor the directed light beam so absorption ofmultiple chromophore in the bioadhesive material is achieved. Forexample, a laser can be tailored to the absorption of both silver andgold nanoparticles in a bioadhesive composition.

In one aspect, the tissue in need of being welded is from a cut or sore.In another aspect, the tissue in need of being welded is from surgery.In an aspect, the tissue in need of welding is from a traumaticdisruption of the tissue.

In one aspect, the bioadhesive composition is suitable for suturing. Forexample, the bioadhesive tissue can be in the form a string or othersuitable suture material for suturing. For example, the bioadhesivecomposition is in the form of a fiber. One singular advantage of thenanocomposites is that it is possible to generate fibers from ELP-basedmaterials using electrospinning, electrospraying and wet-spinningtechniques^([81,85]). PNP-ELP plasmonic fibers (up to 60 μm dia.), forexample which may be generated using wet spinning or 0.2-3 μm usingelectrospinning, may be used for simultaneously suturing the wound andsubsequent LTW for added strength. The bioadhesive composition suturematerial may be used alone or in addition to bioadhesive compositionsdisclosed herein. For example, a bioadhesive suture material may be usedto attach the free ends of the disrupted tissue, and a liquidbioadhesive composition may be used to coat the repair site, or a sheetof bioadhesive composition material may be wrapped around the exteriorof the repair site. The area may then undergo tissue welding to form atight seal at the repair site.

In one aspect, the bioadhesive material can comprise cells. The cells inthe bioadhesive material can promote the connection of tissue upon beingapplied to a site to be welded. Cellularized and non-cellularizedPNP-ELP nanocomposite compositions of the present invention can beemployed for LTW of intestinal tissue alone and in combination withsuturing using known suture material. Fibers of PNP-ELP nanocompositecompositions may be used for simultaneous suturing and laser tissuewelding of tissue, such as colorectal tissue.

In one aspect, the directed light beam is a laser, such as aTitanium-Sapphire laser, Krypton laser, Ruby laser, Chromium dopedchrysoberyl (alexandrite) laser, Divalent samarium doped calciumfluoride (Sm:CaF2) laser, AlGaInP laser, AlGaAs laser, Vertical cavitysurface emitting laser (VCSEL). In one aspect, the wavelength of thelight from the directed light source is in the near infrared. In anotheraspect, the directed light source is manipulated to match the absorptionwavelength of the light absorbing chromophore. For example, the directedlight beam can comprise a wavelength of light that matches theabsorption of gold nanorods. In one aspect, the disrupted tissue iscolorectal tissue. In another aspect, the disrupted tissue can be ablood or lymphatic vessel in the body. For example, the disrupted tissuecan be bowel. In another example, the disrupted tissue can be uniarytract tissue. In another example, the disrupted tissue can be skin. Inanother example, the disrupted tissue can be from a cut, such as asurgical cut.

In one aspect, the directed light beam can be in continuous wavelengthmode. In another aspect, the directed light beam can be in pulsewavelength mode.

In an aspect, the bioadhesive composition further comprises cells.Suitable cells include but are not limited to, NCM460, fibroblasts, stemcells, or mixtures thereof.

In one aspect, the disrupted tissue in need of being welded is from soreor cut. For example, the disrupted tissue in need of being welded isfrom a surgical cut or from a traumatic disruption of a tissue or organ.In one aspect, the disrupted colorectal tissue results from removal of asection of the colon during surgery.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers (e.g., amounts, temperature, etc.), but someerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

Several methods for preparing the compounds of this invention areillustrated in the following Examples. Starting materials and therequisite intermediates are in some cases commercially available, or canbe prepared according to literature procedures or as illustrated herein.

The following exemplary compounds of the invention were synthesized. TheExamples are provided herein to illustrate the invention, and should notbe construed as limiting the invention in any way. The Examples aretypically depicted in free base form, according to the IUPAC namingconvention

A. General Methods

The examples below demonstrate the production of PNP-ELPs suitable forLTW as shown in FIGS. 1A-1C. The PNP-ELP bioadhesive materials withplasmonic nanoparticles, for example gold nanorods and silvernanoparticles, can maximize the efficacy and control of the photothermaleffect. As shown below, the ELP, cross-linked with PNPs, can improve theanastomoses strength and the elasticity of the PNP-ELP nanocompositewill allow for recovery of tissue, such as colon function.

Huang et al. previously generated GNR-ELP nanoassemblies^([2]) andplasmonic nanocomposites^([1]), described in references, each of whichis hereby incorporated herein by reference in its entirety. Huang et al.showed that such nanocomposites possess photothermal properties, supportcell culture, and can be employed for light triggered administration ofhyperthermia and small-molecule drugs^([1]). Additionally, phaseseparation and formation of nanocomposites have been studied. (Langmuir,manuscript accepted for publication, Feburary 2012).

Dallas et al. previously produced silver nanoparticle-ELPnanocomposites. In addition to their plasmonic properties, the knownantibacterial properties^([44]) of silver make them attractive in woundhealing, Dallas et al. is hereby incorporated herein by reference in itsentirety.

The examples below indicate that PNP-ELP nanocomposites can be usedsimultaneously for laser tissue welding, drug (e.g. doxycycline)delivery, and tissue healing in a multifunctional manner. Thus, one canengineer the ELP sequence and modulate nanoparticle type (e.g. GNR orsilver nanoparticle) and composition in order to obtain desiredphysicochemical, mechanical, and photothermal properties.

B. Example 1 Generation of Bioadhesive Compositions

Huang et al. previously generated C_(n)ELPs (C_(n) indicates n number ofcysteines in the ELP repeat sequence) using recursive directionalligation, expressed, and purified as described previously^([2]), whichis hereby incorporated by reference in its entirety. . C2ELPs wereself-assembled on gold nanorods by means of stable gold-thiol bonds,leading to formation of photothermally responsive GNR-C2ELPnanoassemblies or liquid-phase dispersions^([2]). Engineering additionalcysteines (n=8, 12) resulted in phase separation and formation ofGNR-ELP viscoelastic nanocomposites. Briefly, GNR-C₁₂ELP nanoassemblieswere first generated at 4° C. Incubation of these nanoassemblies at 37°C., i.e. above the transition temperature (T_(t)) of C₁₂ELP (T_(t) ofC₁₂ELP=30.4° C.), resulted in temperature-triggered, entropy-dominatedphase transition of the polypeptide, which, in concert with GNR-thiol,and intra- and inter-molecular cysteine-cysteine cross-linking, resultedin the irreversible formation of maroon-colored, plasmonicnanocomposites^([1]) (see, FIG. 2). The nanocomposites werecharacterized using UV-Vis spectroscopy, FTIR, response to NIR laserirradiation, and dark-field imaging in order to investigate theiroptical and photothermal properties (FIGS. 3A-3E)^([1,2]).

The nanocomposites demonstrated a robust photothermal response inresponse to NIR laser due to uniform distribution of GNRs throughout theELP matrix. Small-molecule drugs could be encapsulated in the GNR-ELPnanocomposite. Laser irradiation resulted in release of the drug fromthe nanocomposite, though not wishing to be bound by any particulartheory, it was thought due to structural changes in the ELP at thehigher temperatures brought about by the GNR photothermal effect,indicating that the nanocomposites can be used for laser-triggered drugdelivery.

(1) Mechanical Properties

The PNP content in the bioadhesive compositions can be varied in orderto modulate the mechanical properties, leading to different swellingratios and stiffness of the nanocomposite. Suture materials typicallyposses a tensile strength of ˜130 N/mm^(2[57]) which is much higher thanthe human colon tissue (˜0.4-2 N/mm²)^([58]). Although much stronger,sutures cannot recover the mechanical strength of the ruptured tissue toits original intact value. The mismatch in mechanical strength may bepartly responsible for sub-optimal performance. On the other hand,tensile strength of aortic elastin (˜1 N/mm^(2[59])), synthetic elastin(˜0.2 N/mm^(2[60])), glutaraldehyde cross-linked elastin films (0.3-0.8N/mm^(2[59])) and electrospun tropoelastin fibers (˜0.36 N/mm^(2[61])),are similar to that of the colon. For example, FIG. 4A shows theswelling results for the disclosed bioadhesive materials. Astatistically significant (p<0.001) reduction of swelling ratio(defined: swollen mass (M_(s)) over dry mass (M_(d))) from 12 to 6 wasobserved as the GNR weight percentage increased from 0 to 5.4 wt % at25° C. This is likely due to the cross-linking facilitated by GNRsleading to the formation of rigid network to prevent swelling. Theabsolute shear modulus (|G*|) and loss angle (δ), representing stiffnessand internal energy dissipation of the nanocomposite under dynamicloading, respectively, are investigated as a function of GNRconcentration (see FIGS. 4B and 4C). Both, swelling ratio and stiffnesscan be modulated by altering the composition of PNPs in thenanocomposites. In addition, tensile strength, elastic modulus, strainat failure, and resilience are determined, in order to identify thosecompositions whose mechanical properties match those of the colon.

(2) Proposed Characterization and Photothermal Property Characterizationof PNP-ELP Nanocomposites.

It is typically necessary to heat tissues from 60° C. to 95° C. in orderto achieve maximal anastomosis strength^((26, 39-41, 62]) GNR-ELPnanocomposites was modulated in order to obtain a bulk temperature of˜45° C. following laser irradiation, although the temperature directlyin the path of the laser may be higher. In order to identify materialsthat result in weld temperatures of 65-75° C., the (i.e. GNR or silver),nanoparticle weight % (range: 1-20%) within the ELP matrix, laser powerdensity, and laser irradiation time, can be altered. For example,GNR-ELP, AgNP-ELP, and hybrid GNR-AgNP-ELP nanocomposites were generatedand these nanocomposites absorb light at different wavelengths (seeFIGS. 5A-5C) The temperature can be monitored using a J-thermocouple andan IR camera. An evaluation of mechanical properties (e.g. swellingratio, absolute shear modulus and loss angle) of nanocomposites will becarried out at different temperatures (25-75° C.), in order toinvestigate the integrity of these materials. Any potentialdeformation^([63, 64]) of the plasmonic nanoparticles following laserirradiation can be investigated using Field Emission Scanning ElectronMicroscopy (FE-SEM), TEM and UV-Vis spectroscopy. Optimization of PNPcontent, laser energy and type can be employed to identify conditionswhere the localized temperature is controlled below a shapetransformation threshold, and the temperature near the laser area is˜65-75° C. to avoid tissue shrinkage and discoloration^([65]). Thespatiotemporal temperature distribution following photothermal responseof the nanocomposites can be modeled based on the Pennes bioheatequation using an approach described in Hunag et al. ACS Nano 2010.Stability of PNP-ELP nanocomposites in PBS are evaluated for at leastsix months by monitoring release of nanoparticles (e.g. using NIRabsorbance for gold), and polypeptide fragments (e.g. usinggel-permeation chromatography or GPC) in the supernatant. Differentamounts of doxycycline (100-1000 μg) will be encapsulated anddiffusion-based and laser triggered release of the drug from thenanocomposites are determined as describer^([1]).

C. Example 2 Laser Tissue Welding

GNR-ELP nanocomposites were employed for the ex-vivo laser tissuewelding of porcine intestines. GNR-ELP nanocomposites (10 mm diameter,thickness=247±65 μm) were irradiated fully across with 800 nm CW laserat a rate of 1 mm/sec. This treatment resulted in enhancing themechanical strength (breaking force) of ruptured intestinal tissue to upto 47% of its original intact form (see FIGS. 6A-6C). The results, witha limited parameter set (laser irradiation time, GNR amount, etc.), showthe potential of GNR-ELP nanocomposites for LTW of intestinal/colorectaltissues.

(1) Cellularized PNP-ELP Nanocomposites for Laser Tissue Welding.

Fibroblast-cellularized hydrogels demonstrate collagen deposition^([72])which increases mechanical stiffness^([73]) of the hydrogel compared tothe acellular material. Cellularized nanocomposites facilitate rapidcollagen deposition that imparts mechanical strength to the weldpromoting tissue regeneration. To show this, fibroblast proliferationwas sustained for up to 2 weeks with minimal loss of viability.PEGylated-GNR based nanocomposites showed 55-77% attachment offibroblasts at 24 h compared to tissue culture well plates (control).Encapsulation of 3T3 fibroblasts (see FIGS. 7A-7C) and theirproliferation (see FIGS. 8A-8D) within GNR-ELP nanocomposites wasconfirmed by confocal fluorescence microscopy. It is therefore possibleto cellularize PNP-ELP nanocomposites to promote tissue repair.

(2) Other Experiments

The normal colon epithelial cell line, NCM460^([74]), available fromINCELL Corporation, LLC, are employed for encapsulation within PNP-ELPnanocomposites; fibroblasts and 50-50% co-cultures of the two cell typesare used. Cell viability and proliferation of NCM46 cells from thenanocomposite towards the peripheral areas of the nanocomposites arecharacterized using fluorescence microscopy. If necessary, PNPs arefunctionalized with cationic polymers (e.g. 1,4C-1,4Bis) which werepreviously synthesized^([75, 76]) to promote cell attachment. Asdiscussed above, it was demonstrated that the ability to culture murinefibroblast (NIH 3T3) cells both, on top of as well as insidenanocomposites (prepared using both PEG-modified and unmodified GNRs)with negligible cytotoxicity (see FIGS. 7A-7C).

Cellularized nanocomposites are employed in concert with a scratch woundhealing assay to assess cell migration and proliferation as a healingmodel^([77]). The cellularized PNP-ELP nanocomposite are used as notonly a bioadhesive solder for laser tissue welding of colorectal tissuefor enhanced mechanical strength, but also to facilitate tissueregeneration, mediated by soluble factores^([78, 79]) (FIGS. 8A-8D).Given the role of fibroblast growth factor (FGF), TGF-(3, and EGF inwound healing and repair, ELISA will be used to determine the expressionof growth factors/cytokines^([80]) from the nanocomposites.

Laser irradiation of nanocomposites placed on top of the tissue incisionresults in a fluid-tight seal and enhances the mechanical integrity ofanastomosis. Live cells outside the laser path migrated towardsanastomosis site and growth factors can facilitate tissue regeneration.Both, cell viability outside the direct laser path (green-fluorescentcells) and active migration have been demonstrated using PC3-PSMA humanprostate cancer cells and NIH 3T3 murine fibroblasts, respectively (FIG.8B). In all cases, the bursting pressure, tensile strength ofcellularized scaffolds upon laser tissue welding are compared tonon-cellularized scaffolds, sutured tissue, and the original non-damagedtissue. Collagen and histology staining are carried out as describedpreviously.

Optimized nanocomposites are employed to maximize laser anastomosisstrength of porcine colorectal tissues. Ex vivo fresh tissues arepurchased from Animal Technologies Inc., Texas. Laser-welded tissuesundergo (i) breaking force (tensile strength) measurement using TA XTplus Texture Analyser (Texture Technology Corp., NY) (see FIG. 6A) and(ii) leaking and bursting pressure tests^([29]) to evaluate themechanical integrity and weld strength of the anastomosis site.Specifically, full-thickness incisions (fully/partially across thetissue) are applied on rectangular intestinal sections (1×4 cm) andtubular intestinal sections (˜7 cm in length). The tissues undergobreaking force and bursting pressure testing following LTW. Changes intensile strength, at various time points after LTW, are correlated tohistologic findings. Histologic analyses are performed on the crosssections of repaired tissues, using hematoxylin and eosin (H&E)staining, to evaluate the morphology of collagen, nanocomposite andwound edge^([39]). Conventional suture anastomoses and fibringlue^([71]) will be used as controls.

D. Example 3 Bioadhesive Materials and Properties

(1) Materials

Sodium borohydride, powder, reagent grade, no less than 98.5%,cetyltrimethylammonium bromide (CTAB), 95%, gold (III) chloridetrihydrate (HAuCl₄.3H₂O), +99.9%, L-ascorbic acid, reagent grade werepurchased from Sigma. Crystalline silver nitrate was purchased fromSpectrum and dithiothreitol (DTT) was purchased from EMD. All materialswere used as received without further purification.

(2) GNR Synthesis

Gold nanorods were synthesized using the seed-mediated method asdescribed by El-Sayed et al.^([22]). Briefly, the seed solution wasprepared by adding 0.6 ml of iced-water-cooled sodium borohydride (0.01M) to reduce a solution of 5 ml (0.2 M) of CTAB in 5 ml (0.0005 M) auricacid with vigorous stirring. The growth solution was prepared byreducing 5 ml (0.2 M) CTAB in 5 ml (0.001 M) auric acid containing 280μl (0.004 M) silver nitrate with 70 μl (0.0788 M) L-ascorbic acidsolution. Seed solution (12 μl) was introduced to 10 ml of growthsolution, which resulted in the generation of GNRs after 4 h ofcontinuous stirring. The nanorods were centrifuged once, the supernatantwas removed, and re-suspended in deionized (DI) water to remove extrafree CTAB molecules. This method was employed for generating GNRs thatpossessed absorbance maxima (λ_(max)) in the near-infrared region of thelight absorption spectrum.

(3) Synthesis, Expression & Purification of Cysteine-Containing ELPs

Cysteine-containing ELPs, C₈ELP and C₁₂ELP, were generated via therecursive directional ligation method described previously^([23]). C₈ELPand C₁₂ELP, respectively, contain 8 and 12 cysteine residues in thesequence: MVSACRGPG-[VG VPGVG VPGVG VPGVG VPGVG VPG]₈-[VG VPGVG VPGVGVPGCG VPGVG VPG]_(8(or 12))-WP (SEQ ID NO:1 and SEQ ID NO:2,respectively). Briefly, oligonucleotides encoding the ELPs were clonedinto pUC19 vector, followed by cloning into a modified version of thepET25b⁺ expression vector at the sfiI site. Escherichia coli BLR(DE3)(Novagen) was used as a bacterial host. Both C₈ELP and C₁₂ELP wereexpressed, purification lyophilized and stored at room temperature, asdescribed previously^([23]).

(4) Determination of Transition Temperature

The transition temperatures (T_(t)) of C₈ELP and C₁₂ELP werecharacterized by monitoring the absorbance at 610 nm as a function oftemperature with an UV-visible spectrophotometer (Beckman DU530) in 0.5×phosphate-buffered solution (PBS). Briefly, 1 ml of C₈ELP (0.5 mg/ml in0.5×PBS) and 1 ml of C₁₂ELP (1 mg/ml in 0.5×PBS) were prepared andplaced in 1.5-ml disposable cuvettes. The temperature of C_(n)ELP (n:the number of cysteines in the ELP repeat sequence; n=8/12) was tuned byplacing the C_(n)ELP-contained cuvette into a Precision 288 DigitalWater Bath (Thermo Scientific) and was recalibrated by FLUKE 54 II (TypeK) thermometer before absorbance measurement. The absorbance of C_(n)ELPwas monitored at 610 nm with an UV-visible spectrophotometer (BeckmanDU530) immediately after withdrawing the cuvette out of the water bath.The T_(t) is defined as the temperature at which the absorbance ofC_(n)ELP solution reaches 50% of the maximum value. The temperatureresponse of the C₈ELP and C₁₂ELP indicated T_(t) values of 31.3 and30.5° C., respectively.

(5) Generation of C_(n)ELP-GNR Nanoassemblies

Two different ELPs, C₈ELP and C₁₂ELP, containing 8 and 12 cysteines inthe ELP repeat sequence, respectively, were employed in the currentstudy. Cysteine-containing ELPs were self-assembled on GNRs (CTAB-GNRs)whose peak absorbance (λ_(max)) was at 800 nm. ELPs were self-assembledon GNRs overnight at 4° C., leading to formation of the nanoassemblies(C_(n)ELP-GNR assemblies) via gold-thiol bonds. Briefly, 1 ml ofC_(n)ELP (2 mg/ml in 1×PBS) was mixed overnight with 1 ml of GNR(optical density at 800 nm=0.5) dispersion in DI water at 4° C. to forma 2-ml C_(n)ELP-GNR dispersion (1 mg/ml in 0.5×PBS). Prior toself-assembly, 20 mg of reductacryl resin (EMD Biosciences, Inc.) wasadded to ELP (1 ml) solution for 15 min in order to reduce the cysteinesin the polypeptide^([24]). Reduced ELP was separated from the resin bycentrifugation at 13,000 rpm for 10 min, and immediately added to GNRsat a volumetric ratio of 1:1 and stirred overnight at room temperature.Equivalent concentrations of GNRs (without self-assembled C_(n)ELP) andC_(n)ELP (without GNRs) were used as controls in the experiments.C_(n)ELP (2 mg/ml in 1×PBS) was added into DI water at a 1:1 volumeratio, to form C_(n)ELP solution (1 mg/ml in 0.5×PBS; C_(n)ELP alone).GNRs in DI water (1 ml; λ_(max)=800 nm; optical density at 800 nm=0.5)were added to an equal volume (1 ml) of 1×PBS in order to bring thefinal concentration to 0.5×PBS (GNR alone).

(6) Formation of C_(n)ELP-GNR Matrices

A volume of 1 ml C₈ELP-GNR, and C₁₂ELP-GNR solutions (1 mg/ml, 0.5×PBS,optical density: 0.25), in 1.5-ml microcentrifuge tubes, was incubatedin a 37° C. water bath for 6 h in order to allow the phase separation ofGNR-C_(n)ELP, resulting in the formation of the matrix at the bottom ofthe tubes. The matrices were subsequently cooled and stored at roomtemperature. GNRs (without self-assembled C_(n)ELP) and C_(n)ELP(without GNR) solutions were used as controls in the experiment. Tostudy the kinetics of matrix formation, the absorption spectra of thesupernatant of both C₈ELP-GNR and C₁₂ELP-GNR dispersions were determinedat different times using a temperature-controlled plate reader (BiotekSynergy 2) during water heating (bath incubation) and cooling. Thespectra were typically measured between 300 and 999 nm. The C₈ELP-GNRand C₁₂ELP-GNR matrices are stable at room temperature for at least 1month.

(7) Dissolution of C₈ELP-GNR Matrices

For dissolution experiments, the PBS supernatants were removed fromindividual C_(n)ELP-GNP matrices and replaced with equivalent volumes of10 mM dithiothreitol solution for 30 min at 4° C., following which,absorbance spectra were determined as a function of time in order toinvestigate dissolution kinetics.

(8) Fourier-Transform Infrared Spectroscopy

Gold nanorods and C₈ELP- and C₁₂ELP-based matrices were loaded on agermanium-attenuated total reflectance crystal, such that they coveredthe central area of the crystal. The sample chamber was equilibrated toapproximately 4 mb pressure in order to minimize the interference ofatmospheric moisture and CO₂. The absorption spectrum was measuredbetween 650 and 4,000 cm⁻¹ using a Bruker IFS 66 v/S FT-IR spectrometerand the background spectrum was subtracted from all sample spectra, asdescribed previously^([25]).

(9) Field-Emission Scanning Electron Microscopy

Scanning electron microscopy (SEM) samples were prepared by placingC_(n)ELP-GNR matrices on a flat alumina substrate. The matrix on thesubstrate was allowed to dry out in open laboratory atmosphere. SEMimages were obtained with an environmental field-emission SEM (PHILIPSFEI XL-30 SEM) operating an accelerating voltage of 25 kV, and severalmagnifications between 2500 and 20,000×.

(10) Photothermal Properties of a C₁₂ELP-GNR Matrix Film

C₁₂ELP-GNR dispersion (750 μl of 1 mg/ml in 0.5×PBS; optical density:0.25; 4° C.) in 1-mm diameter acrylic cell (homemade) was immediatelyincubated in 37° C., 5% CO₂ environment for 3 h, in order to allowmatrix formation on top of a tissue culture-treated 1.5-mm diametercover slip originally placed at the bottom of the acrylic cell. Thesupernatant was removed from the acrylic cell after incubation and theabsorption spectrum of the C₁₂ELP-GNR film was determined using a platereader (Biotek Synergy 2) at room temperature. The spectrum was measuredbetween 300 and 999 nm at five individual times.

The photothermal properties of the matrix were determined usingirradiation with a titanium CW sapphire laser (Spectra-Physics, Tsunami)pumped by a solid-state laser (Spectra-Physics, Millennia). Briefly, theexcitation source was tuned to 850 nm in order to coincide with thelongitudinal absorption maximum of the C₁₂ELP-GNR matrix. The C₁₂ELP-GNRmatrix was placed at the bottom of a 24-well plate (Corning) with 500 μlof 1×PBS as the supernatant over the matrix. The well was irradiatedwith laser light at 850 nm at power densities of 20 or 25 W/cm² for 15min, and the dispersion temperature was monitored by FLUKE 54 II (TypeK) thermocouple during laser exposure. Controls with only 500 μl of1×PBS solution in 24-well plates (i.e., without C₁₂ELP-GNR film) werecarried out; temperature remained invariant at 24±0.5° C. after 15 minlaser exposure in this case.

(11) Formation of 17-AAG-Loaded C₁₂ELP-GNR Matrix Film (24-Well Plate)

C₁₂ELP-GNR dispersion (750 μl of 1 mg/ml, 0.5×PBS, optical density: 0.25at 4° C.), containing 750 μg of 17-AAG (LC Laboratories, MA, USA) wasplaced in the 1-mm diameter acrylic cell, and immediately transferred toan incubator (37° C., 5% CO2) for 3 h, allowing phase separation andformation of 17-AAGloaded C₁₂ELP-GNR (17-AAG-C₁₂ELP-GNR) matrix on topof a tissue culture-treated 1.5-mm diameter cover slip. While 6 h werepreviously employed for generating C₁₂ELP-GNR matrices (without 17-AAG),analysis of matrix formation kinetics indicated that 3 h were sufficientto generate the matrix. As a result a 3-h incubation period was used forgenerating 17-AAG-C₁₂ELP-GNR matrices in order to reduce processingtimes. Following incubation, the supernatant, containing free 17-AAGmolecules, was removed from the acrylic cell after 3 h, and assayed forconcentration using absorbance analysis. The amount of 17-AAGencapsulated in the matrix was determined from a mass balance on thedrug. Briefly, absorbance values of known concentrations of 17-AAG at335 nm were employed to generate a standard calibration curve. Followingmatrix formation, the concentration of 17-AAG in the supernatant wasthen back-calculated based on the absorbance and the calibration curve.Since the initial amount of 17-AAG is known, the amount encapsulated inthe matrix was calculated as the difference of 17-AAG before and afterencapsulation. The absorption spectrum of the 17-AAG encapsulatedC₁₂ELP-GNR film was determined at room temperature using a plate reader(Biotek Synergy 2) with five individual measurements. A peak at 335 nmwas used to detect encapsulation of the drug.

(12) Release of 17-AAG from 17-AAG-C₁₂ELP-GNR Matrices

Drug (17-AAG)-loaded C₁₂ELP-GNR matrices were prepared as describedabove and placed in a 24-well plate with 500 μl of 1×PBS. Thediffusional release of 17-AAG from the matrix was monitored for 24 h.The laser beam was tuned to 2 mm in diameter for all near-infraredirradiation-triggered drug-release studies. The first laser irradiationlasted for 5 min (850 nm, 25 W/cm²). Five subsequent laser irradiations(850 nm, 25 W/cm²) lasted for 10 min, followed by a 20-min periodwithout laser irradiation each. The temperature profile during the10-min laser exposure was monitored using a K-type thermocouple.

(13) Cell Culture

The PC3-PSMA human prostate cancer cell line^([26]) came from theMemorial Sloan Cancer Center (NY, USA). RPMI 1640 with L-glutamine andHEPES (RPMI-1640 medium), pen-strep solution: 10,000 units/ml penicillinand 10,000 μg/ml streptomycin in 0.85% NaCl, and fetal bovine serum(FBS) were purchased from Hyclone. Serum-free medium is RPMI-1640 mediumplus 1% antibiotics. Serum-containing medium is serum-free medium plus10% FBS. Cells were cultured in a 5% CO2 incubator at 37° C. usingRPMI-1640 medium containing 10% heat-inactivated FBS and 1% antibiotics(10,000 units/ml penicillin G and 10,000 μg/ml streptomycin).

(14) Cell Culture & Laser Irradiation on C₁₂ELP-GNR Matrices

C₁₂ELP-GNR and 17-AAG-C₁₂ELP-GNR matrices on tissue culture cover slipswere prepared as described previously. Prior to cell culture, thematrices were pretreated with 500-μl serum containing media in a 24-cellculture well plate (Corning) overnight in order to promote cellattachment. The serum-containing media was removed after incubation andthe matrixcoated cover slips were washed twice with freshserum-containing media. PC3-PSMA human prostate cancer cells were seededon top of the matrices in several wells with a density of 150,000cells/well and allowed to attach for 24 h at 37° C., in a 5% CO₂incubator. For the laser irradiation experiment, the excitation sourcewas tuned to 850 nm in order to coincide with the longitudinalabsorption maximum of the C₁₂ELP-GNR film. Matrices with PC3-PSMA cellswere exposed to laser irradiation at 850 nm at a power density of 25W/cm² for 7 min (no laser exposure for the control samples). Thesolution temperature was monitored by a FLUKE 54 II (Type K)thermocouple during laser exposure. Fluorescence-based Live/Dead® assaywas employed to investigate cancer cell viability 24 h after laserirradiation. Briefly, cells were treated with 4 μM ethidium homodimer-1(Invitrogen) and 2 μM calcein AM (Invitrogen) for 30 min, and imagedusing Zeiss AxioObserver D1 inverted microscope (10×X/0.3 numericalaperture objective; Carl Zeiss MicroImaging Inc., Germany). Dead/dyingcells with compromised nuclei stained positive (red) for EthD-1,viable/live cells stained green for calcein AM.

(15) Results and Discussion

Cysteine-containing ELPs (CnELPs; n=8 or 12, indicating 8 or 12cysteines in the ELP repeat sequence) were synthesized via recursivedirectional ligation, expressed in E. coli, and purified, as describedpreviously^([23]). The transition temperatures (T_(t))^([27]) of C₈ELPand C₁₂ELP were determined to be 31.3 and 30.5° C., respectively.CTAB-GNRs, with maximum peak absorbance at 800 nm in the near-infraredregion of the absorption spectrum, were generated using theseed-mediated growth method^([22]). The cysteines in CnELPs were firstreduced using Reductacryl®^([24]), following which they were employed tofacilitate the self-assembly of polypeptide molecules on GNRs at 4° C. Ared-shift of approximately 20 nm (from 800 to 820 nm) was observed inthe maximal absorbance peak, which indicated the formation of CnELP-GNRnanoassemblies^([23]).

The temperature transition property of C_(n)ELPs was exploited forgenerating C₈ELP-GNR and C₁₂ELP-GNR matrices from C_(n)ELP-GNRnanoassemblies. C₈ELP-GNR and C₁₂ELP-GNR nanoassemblies were kept at 37°C. (>T_(t) for both ELPs) for 6 h. Incubating GNRs and C_(n)ELPs belowthe transition temperature results in the formation of well-dispersed‘assemblies’. However, incubation at temperatures above T_(t) results intemperature-triggered, entropy-dominated phase transition ofELP^([27, 28]), in addition to GNR-thiol (from ELP cysteines), andintra- and inter-molecular cysteine-cysteine crosslinking resulting inthe formation of reddish-colored plasmonic matrices (FIGS. 9A-9F).Lowering the temperature below T_(t) did not result in dissolution ofthe C₈ELP-GNR and C₁₂ELP-GNR matrices (FIG. 9D), which indicated thatmatrix formation was not reversible with temperature, owing to extensivecrosslinking. Unlike C_(n)ELP-GNR matrices, C_(n)ELP in the absence ofGNRs (FIG. 9G) demonstrated a reversible phase transition process(change in optical density), but no matrix formation. GNRs, in theabsence of C_(n)ELP, showed no visible differences following thetemperature changes (FIG. 9H). Kinetics of matrix formation werefollowed using the light absorption spectrum of the C_(n)ELP-GNRsupernatant. Matrix formation was not observed immediately after takingthe GNR-C₈ELP and GNR-C₁₂ELP nanoassemblies out of the 4° C. cooler; thelight absorption spectrum at time=0 min in FIG. 2 shows a profilecharacteristic of the GNRs in the dispersion. The maximum opticaldensity was 0.25 for both C₈ELP-GNR and C₁₂ELP-GNR dispersions underthese conditions. Incubation of the C₈ELP-GNR and C₁₂ELP-GNR assembliesat 37° C. first resulted in an increase in optical density indicatingaggregation of the GNR-CnELP nanoassemblies above the T_(t) of therespective C_(n)ELPs; the optical densities of C₈ELP-GNR and C₁₂ELP-GNRassemblies were highest 15 min after incubation. Further incubation ledto crosslinking and phase separation of the C₈ELP-GNR and C₁₂ELP-GNRmatrices, leading to precipitation of the solid-phase matrix.Precipitation from the liquid dispersion resulted in the sequestrationof both GNRs and the ELP in the solid phase matrix. This, in turn, ismanifested as a decrease in optical density or absorbance of thesupernatant (FIG. 10).

Fourier-transform infrared spectroscopy for C₈ELP-GNR and C₁₂ELP-GNRnanocomposites indicated a combination of N—H bending and C—N stretchingvibrations (amide II peak) at wave number 1550 cm⁻¹, a peakcorresponding to the C═O stretches in the amide functionality (amide Ipeak) at 1645 cm⁻¹, and a band corresponding to the N—H stretchingvibrations, at 3200 and 3000 cm-1 (FIG. 12), which are characteristic ofELP spectra^([29]). Field emission SEM images showed that GNRs (˜50 nmin length) were well dispersed in the matrices (FIG. 13), indicating thepossibility that the matrices were able to demonstrate stableplasmonic/photothermal properties. The absorbance spectrum of theC₁₂ELP-GNR matrix (FIG. 14) showed the transverse (520 nm) andred-shifted longitudinal peak (850 nm) characteristic of GNRs,indicating that the matrices indeed demonstrated plasmonic propertiesdue to the uniform distribution of the GNRs. Photothermal properties ofC_(n)ELP-GNR matrices were investigated by recording the temperature ofPBS supernatant (500 μl) above the matrix in 24-well plates.Temperatures in each case reached their respective steady-state values(˜46° C.) 5 min following laser irradiation, consistent with ourprevious observations with GNRs^([23,30,31]); the steady-statetemperatures did not change following the relatively minor change inpower density from 20 to 25 W/cm².

Suboptimal administration of hyperthermia can result in the incompleteablation of tumors and selection of clones that are resistant totreatment. While temperatures above 46° C. result in significant loss ofcell viability, mild or moderate hyperthermic temperatures (40-46° C.)can have differential cytotoxic effects on cells, leading to variableefficacies. Constitutive and induced expression of HSPs, includingHSP90, results in the refolding of proteins denatured by hyperthermiaand, therefore, results in overcoming the apoptotic effects of thetreatment. In particular, HSP90 is a stress-related protein, whichinteracts with several client proteins and regulates key processesinside cells, including protein degradation, and aids cancer cellsurvival following hyperthermia. Strategies that combine hyperthermicablation with chemotherapeutic drugs that can overcome HSP-inducedresistance can result in enhanced efficacy of hyperthermia as anadjuvant treatment. As a representative example of this approach, thechemotherapeutic HSP90 inhibitor was incorporated in 17-AAG in thematrix, for generating a multifunctional matrix capable ofsimultaneously administering both hyperthermia and chemotherapy, inorder to enhance the ablation of cancer cells. The HSP90 inhibitor wasincorporated within C₁₂ELP-GNR matrices during their formation leadingto 17-AAG-C12ELP-GNR matrices (FIG. 15). The absorbance spectrum of17-AAG-C₁₂ELP-GNR matrices demonstrated an additional peak at 335 nm(FIG. 15), which was indicative of the incorporation of 17-AAG withinthe polypeptide matrix; approximately 550 μg of the drug wereincorporated within a single matrix.

FIG. 16 shows a representative release profile of the 17-AAG from thematrix. The matrix was first placed in PBS to investigate diffusionalleaching of the drug. Approximately 10 μg of the drug was released inthe first hour, following which an additional 7 μg of the drug werereleased over the next 23 h, indicating that only a total of 3% of theencapsulated drug leached out due to diffusion. This demonstrates thatthe matrices are able to stably incorporate chemotherapeutic drugs withminimal loss owing to leaching. This is significant, since unintendeddrug loss from the matrix can result in undesired side effects.Subjecting the matrix to laser irradiation resulted in an increase inlocal temperature due to the photothermal effects of GNRs, which, inturn, led to enhanced release of 17-AAG from the matrix, presumably dueto ELP structural changes and aggregation above the transitiontemperature, which in turn, can result in contraction of the matrixleading to drug efflux. The concomitant temperature increase is shown inFIG. 16 and is similar to that observed with the matrix in the absenceof the drug (FIG. 14). A 5-min laser irradiation pulse (850 nm laser, 25W/cm²) resulted in the release of only 2.5 μg 17-AAG, indicating longerexposure times were necessary for increased release of the drug.Subsequent laser exposures were, therefore, carried out for 10 min each(850 nm, 25 W/cm2), which resulted in the release of 12.6 μg±2.4 μg foreach round of laser irradiation. Discolored spots on the C12ELP-GNRmatrix (FIG. 16) indicate regions of drug release following exposure tothe laser. This is consistent with the temperature profiles in FIG. 16,which indicated that merely reaching hyperthermic temperatures may notbe enough to trigger drug release and that sustained hyperthermictemperatures are required.

In order to further investigate the role of laser-induced drug release,the matrix for diffusional drug release was first investigated, followedby incubation at moderately hyperthermic temperatures (42° C.), andfinally laser treatments. The total amount of drug originallyencapsulated in this matrix was approximately 614 which was higher thanthe amount encapsulated in the matrix shown in FIG. 16. As seen in FIG.16, higher 17-AAG quantities were released following laser irradiationcompared with the water bath incubation treatment. It is possible thattemperatures directly in the path of the laser are significantly higherthan 42-44° C., and lead to greater drug release due to more significantchanges in the matrix at these locations.

Taken together, these results indicate that the photothermal propertiesof the polypeptide matrix facilitate local increases in temperaturefollowing laser irradiation, which in turn triggers release of theencapsulated drug, presumably due to a combination of increased drugdiffusivity and ELP aggregation and contraction at temperatures abovethe polypeptide transition temperature.

The efficacy of the simultaneous was then tested through administrationof hyperthermia and HSP90 inhibitor for the ablation of prostate cancercells. In order to account for the efficacy of this combinationtreatment, two ‘single-agent’ treatments were first carried out:hyperthermia alone, in which the matrix without the 17-AAG drug wasemployed for killing cancer cells only due to hyperthermic temperaturesin the absence of the drug, and 17-AAG alone, in which loss of cancercell viability due to constitutive 17-AAG diffusional release from thematrix was evaluated in the absence of laser-induced hyperthermia.

C₁₂ELP-GNR matrices (without 17-AAG) supported the growth of PC3-PSMAhuman prostate cancer cells, indicating that the plasmonic matrix wasnot toxic to cells. For the ‘hyperthermia alone’ treatment, cells wereirradiated with an 850 nm laser (25 W/cm² laser for 7 min) and cellviability was determined using the Live/Dead assay 24 h after the lasertreatment. Phase contrast and fluorescence microscopy images wererecorded immediately after staining. As expected, laser irradiationresulted in significant death of PC3-PSMA cells directly in the path ofthe laser beam as seen from the red-stained cells in FIG. 17, consistentwith previous observations in the literature^([32,33]). However, cellsoutside the path of the laser beam did not undergo any loss of viabilityas seen from the green-stained living cells in FIG. 17. These resultshighlight spatial limitations associated with nanoparticle-mediatedhyperthermic ablation of cancer cells; while nanoparticles and laserirradiation can be employed for localized treatments, effectivetreatment can be administered only over a limited region, leading toineffective treatment. Importantly, the plasmonic matrix isbiocompatible and can be used for the hyperthermic ablation of cancercells, and in treatments where the spatial limitations of hyperthermiaare not a concern.

C₁₂ELP-GNR plasmonic matrices, containing the anti-HSP90 drug 17-AAG(17-AAG-C₁₂ELP-GNR), were evaluated in the absence of laser-inducedhyperthermia (drug-alone treatment). The matrices were able to supportcell culture for 48 h, indicating that the constitutive diffusionalrelease of 17-AAG from the matrix was not sufficient to induce celldeath in cancer cells (FIG. 18). In order to investigate the efficacy ofthe combination treatment for killing cancer cells, PC3-PSMA cells wereexposed to an 850-nm laser (25 W/cm² laser for 7 min) and as before,cell viability was evaluated 24 h following the laser treatment. While asingle circular shape corresponding to the size of the laser beam couldnot be located in this case, dead cells could be seen throughout thematrix (FIG. 19), indicating that a combination of the hyperthermictemperatures and the triggered release of the anti-HSP90 drug 17-AAG wasresponsible for extensive cancer cell death. The synergistic actionbetween these combination treatments is demonstrated by quantitativeanalysis of the cell death results (FIG. 20). Drug-alone and laser-alonetreatments resulted in minimal loss of cell viability (<10% of the cellpopulation). However, the combination treatment (laser-inducedhyperthermia and release of the HSP90 inhibitor) resulted in over 90%loss in cell viability (FIG. 20). The results indicate that drugloadednanoparticle-polypeptide matrices can simultaneously overcome cancercell resistance to nanoparticle-induced hyperthermia, which is mediatedby HSP overexpression, and spatial limitations of laser-inducedhyperthermia using plasmonic nanoparticles (Table 1).

Table 1

TABLE 1 Summary of combination treatments. Laser power density Celldeath C_(n)ELP-GNR matrices (7-min irradiation) Laser spot PeripheryC₁₂ELP-GNR  0 W/cm² No No C₁₂ELP-GNR 25 W/cm² Yes No 17-AAG-C₁₂ELP-GNR 0 W/cm² No No 17-AAG-C₁₂ELP-GNR 25 W/cm² Yes Yes 17-AAG:17-(allylamino)-17-demethoxygeldanamycin; ELP: Elastin-like polypeptide;GNR Gold nanorod.

Example 3 demonstrates that engineered polypeptides can be interfacedwith GNRs, resulting in the formation of stable, degradable andbiocompatible plasmonic matrices.

E. Example 4

Nanocomposite preparation procedure: Gold nanorods (0.24 mM), inpresence of CTAB concentrations less than 0.25 mM, were incubated withC12ELP overnight at 4° C., leading to formation of the nanoassemblies(dispersion) mediated by gold-thiol bonds. Briefly, prior toself-assembly, C12ELP was treated with Reductacryl® resin for 15 min ata 1:5 weight ratio, in order to reduce the cysteines in the polypeptidechain. Reduced C12ELP (2 mg/ml in 1× phosphate buffered-saline or PBS)was separated from the Reductacryl® resin by centrifugation at 8,000 rcffor 10 min and mixed with GNR dispersions at a volumetric ratio of 1:1.Solid-phase nanocomposites were prepared by incubating 1.5 ml C12ELP-GNRnanoassemblies (dispersion) in a 1 cm diameter home-made acrylic columndevice at 37° C. (or 60° C.) for 4 h. This led to temperature-triggered,entropy-dominated phase transition of C12ELP, which, in concert withGNR-thiol and intra- and inter-molecular cysteine-cysteinecross-linking, resulted in the formation of maroon-colored plasmonicnanocomposite film on a circular cover slip originally placed at thebottom of the device.

F. Example 5

Tissue preparation: Tissue samples were defrosted overnight at 4° C.following which they were kept moist with 1×PBS at 25° C. for lasertissue welding. A 5 mm full thickness incision was applied at the centerof the intestine section (4×1 cm, ˜0.1 cm thick). The incision edgeswere brought into contact with one another, nanocomposite (1 cmdiameter) was applied on top of the serosa layer and across the incisionwith full contact. Laser irradiation (20 W/cm²) was applied verticallyat a speed of 1 mm/second across the nanocomposite for 1, 3, 5, and 7minutes and samples were kept moist during welding to minimize charring.

Cell Culture on Nanocomposite Solders: NIH 3T3 murine fibroblast cellswere cultured at 5% CO2 and 37° C. using DMEM medium containing 10%heat-inactivated fetal bovine serum and 1% antibiotics. Thebiocompatibility of nanocomposites containing various GNR (1.9-5.4 wt %)and PEG (0-19.7 wt %) weight percentages was evaluated in 96-wellplates. Nanocomposites were formed at the bottom of the wells andtreated with serum-containing cell culture medium. Fibroblasts (5,000cells/well) were seeded on top of nanocomposites for 24, 48 and 72hours. Cell viability analyses were carried out using thefluorescence-based LIVE/DEAD® assay (Invitrogen) and Zeiss AxioObserverD1 inverted microscope (Carl Zeiss Microlmaging Inc.). Quantitativeanalysis was carried out by counting cells using the ImageJ software.

Laser Tissue Welding: A titanium sapphire laser pumped by a solid-statelaser (Spectra-Physics, Millennia) was employed for laser tissuewelding. The excitation source (continuous wave, 2 mm beam diameter) wastuned to overlap with the λ_(max) of the nanocomposites at 800 nm.Tissue samples were defrosted in Nanopure water and kept moist at 25° C.for laser tissue welding.

Tensile Strength Measurements: An 8 mm full thickness incision wasapplied at the center of the intestine section (4×1 cm, ˜0.1 cm thick).The incision edges were brought into contact with one another,nanocomposite (1 cm diameter) was applied on top of the serosa layer andacross the incision with full contact. Laser light (20 W/cm²) wasapplied vertically at a speed of 1 mm/second across the nanocompositefor 60 seconds, and samples were kept moist during welding to minimizecharring. After welding, tissue tensile strength was measured using TAXT plus Texture Analyser (Texture Technology Corp., NY) with a 5 kg loadcell. Welded tissues were held with pneumatic grips to prevent slippingduring testing. Testing was carried out in the tension mode at a rate of0.5 mm/second until failure. The maximum force (N) achieved before thetissue breakage was recorded and reported in ultimate tensile strength(UTS, kPa). Intact porcine small intestine sections were subjected tomechanical testing to determine the UTS of uncut specimens. Datareported represent the mean±one standard deviation from at least threeand up to twelve individual samples.

Bursting and leaking pressure tests were conducted on tubular porcineintestines. A home-made pressure detection system was designed andbuilt. The tubular porcine intestines were cut into approximately 10 cmsections, leaving both ends opened. A full thickness incision (˜5 mm)was applied to the center of the tubular intestine. The nanocompositewas applied to the incision. The CW laser (20 W/cm²) was then applied tothe nanocomposite (GNR 5.4 wt %) and tissue for various durations (1, 3,5, and 7 min). After LTW was complete, the intestines were tightlyclamped at both ends. A 21G Precision Glide needle was inserted into thetissue and dyed water was fed into the intestine sections. The pressurewas monitored and recorded at the leaking and bursting points. Theleaking pressure was defined as when the first drop of colored water wasseen coming out of the weld site[1]. The bursting pressure was definedas when a stream of water was seen coming out of the weld site. Controlbursting and leaking pressure tests were conducted on intact and cuttissues. The bursting pressure site was always along the length of theintestine. Dye leakage from the needle puncture site was considerednegligible.

Bacteria Leakage Study: The leakage of Escherichia coli DH5-α bacterialcells from intestines was evaluated. A 5 mm incision was applied to thecenter of each 10 cm tubular intestine and subjected to differenttreatments. Immediately after treatment, the tubular intestines werehung vertically in Erlenmeyer flasks (each filled with 190 mL of freshLB broth) leaving two open ends pointing up. The U-shape hanging methodensures the incision (or welded) sites were submerged into to the freshLB broth. A 10 mL culture of bacterial cells at an optical density(OD₆₀₀) of 0.5 were placed inside the intestine and allowed incubation(37° C., 100 rpm). The optical densities of the fresh LB broth weremonitored as a function of time as an indication for leakage.

Thermal Imaging: Following tissue preparation, tissue were images withan IR camera (FLIR s60) immediately before laser welding. A 5 cm pieceof plastic kept on ice was placed next to the tissue for reference.During laser welding, IR images of the sample were taken at 30 secondintervals and immediately after welding were completed. Followingwelding, the ELP-GNR matrix solder was removed from the tissue, and baretissue was also imaged.

Histology. Immediately following welding, ELP-GNR matrix solders wereeither removed from tissue or left in place. Tissue samples were washedonce in 1×PBS and fixed by immersion in Zamboni's fixative. Zambonifixative is neutral buffered formalin made 0.18% with picric acid. Thespecimens were dehydrated through an increasing ethanol gradient,cleared with toluene, and embedded in Paraplast+ at 60° C. Tenmicrometer thick sections were cut on an AO rotary microtome, collectedon glass slides, and stored overnight at 40° C. The wax sections weredeparaffinized with toluene and brought from ethanol to Nanopure™ watergradually. The sections were stained with hematoxylin and eosin (H&E)according to the manufacturer's instructions, dehydrated once more, andmounted in Permout. Micrographs were collected with an inverted Nikonmicroscope equipped with an Olympus DP25 color camera.

Collagen-C₁₂ELP-GNR Nanocomposites. 5.4% GNR-wt % Collagen-C₁₂ELP-GNRNanocomposites were synthesized similarly as explained above.Nanocomposites were synthesized at ratios of 75%-25% ELP-Collagen. Forexample, at a 75%-25% C₁₂ELP-collagen, C₁₂ELP (1.5 mg) was co-incubatedwith GNRs (115 μg) at 4° C. overnight. Following formation ofC₁₂ELP-GNRs, the nanoassemblies were centrifuged at 6000 rcf andre-dispersed in 100 of Rat Tail Type 1 Collagen Solution (5 mg/ml).Collagen—GNR-C₁₂ELP nanoassemblies mixtures were placed in a device andincubated at 37° C. overnight, leading to the formation ofCollagen-C₁₂ELP-GNR nanocomposites on top of a glass coverslip.

(1) Results

Tensile strength. Cellularized as well as non-cellularizednanocomposites were investigated as solders for laser-based welding ofporcine small intestines ex vivo. The injury model employed in thisstudy is representative of bowel tissue after conventional anastamoseswith leakage. Following an injury to the intestine, the plasmonicnanocomposite (1 mm diameter and ˜2 mg) was applied to the incision,followed by laser treatment. The tensile strength of the rectangulartissue section was employed to evaluate the mechanical integrity ofdifferent treatments, see FIG. 6A. As expected, ruptured and intactsmall intestine sections possess the lowest (0.11±0.01 MPa) and highest(0.45±0.02 MPa) ultimate tensile strengths, respectively, see FIG. 6B.In the absence of the plasmonic nanocomposites, laser irradiation alone(20 W/cm², 1 mm/sec and 3 min) across the incision did not enhance thetensile strength of the ruptured intestine. In absence of laserirradiation, nanocomposites alone demonstrated negligible adhesion, andenhanced the tensile strength of the ruptured tissue by a modest ˜0.03MPa (p=0.052, n=11).

NIR laser irradiation (20 W/cm²; constant speed of 1 mm/sec) ofnanocomposites containing 1.9, 5.4 and 8.7 wt % GNRs resulted in bulktemperatures of 46±1.1, 61±1.5 and 64±0.9° C. respectively (n=9), due tothe photothermal properties of these plasmonic biomaterials. It islikely that the temperature at the site of the weld may be much higherthan the bulk temperature. Irradiating ruptured nanocompositescontaining 1.9 wt % and 5.4 wt % GNR with NIR laser for only one minuteresulted in an increase in the ultimate tensile strength up to 0.17±0.01MPa and 0.22±0.01 MPa, respectively. The higher recovery in case of GNRconcentration of 5.4 wt % may be due to the higher welding temperature61±1.5° C. attained in this case. It is typically necessary to heattissues above 60° C. in order to induce coagulation of proteins forobtaining robust welds[2, 3]. Increasing the laser irradiation time from1 minute to 7 minutes, and increasing the GNR content in nanocompositesfrom 5.4 to 8.7 wt % did not enhance the tensile strength of the weldedtissue further. Standard suturing techniques allow for up to 60%recovery of the mechanical strength of ruptured bowel intestinal tissueby 3 to 4 days[4, 5]. It was demonstrated that laser treatment incombination with nanocomposites can enhance the tensile strength ofruptured intestinal sections up to approximately 47% of the originalintact form.

Fibroblast-cultured nanocomposites were also used for welding theruptured intestine, see FIG. 6C; fibroblasts were cultured on top of thenanocomposites (GNR 1.9 wt %) for 1, 4, and 7 days before laser tissuewelding. In all cases, welding strengths were similar to those observedwith a cellular nanocomposites, indicating that materials cellularizedwith judicious choice of cells can further participate in repair andregeneration of welded tissues.

A critical aspect of sealing intestinal and colorectal tissues involvesprevention of leakage of luminal fluid after anastomosis. Exposure ofsurrounding tissues to this bacteria-rich fluid can result in sustainedinflammation, shock, and mortality[6-8]. To ensure thatnanocomposite-assisted laser tissue welding results in fluid-tightsealing, the following were investigated: (i) the leakage and burstingpressure (defined in the experimental section) and (ii) bacterialleakage following welding.

Bursting and Leaking Pressure. Nanocomposites (˜2 mg), at a fixed GNRconcentration of 5.4 wt %, were first applied to the 5 mm cut, followedby laser irradiation leading to temperature increase up to 61±1.5° C.The leakage and bursting pressures were measured immediately afteranastomosis using a device and reported in pounds per square inch (psi),see FIG. 21. As expected, the ruptured and intact intestine demonstratedthe lowest and highest leakage/bursting pressures, respectively. In caseof ruptured intestines, bursting was observed immediately and wasfollowed by leakage. Both, the leakage and bursting pressures wereapproximately 0.2 psi. In case of intact intestines, the first evidenceof leakage was observed at the needle piercing site at a pressure of 7.2psi, while bursting was observed along the tissue when the pressurereached 12 psi. Ruptured intestines treated with laser alone (withoutnanocomposite) and nanocomposite alone (without laser treatment)demonstrated negligible leakage/bursting pressures (<1 psi), indicatingthat these treatments had minimal effect on repair. Laser irradiation ofthe nanocomposite at the incision site increased both the tissue leakageand bursting pressures. Increasing laser irradiation time from 1 to 7minutes resulted in an increase in both, leakage and bursting pressuresto up to 2.8 and 5.8 psi, respectively. In these cases, burstingimmediately followed leakage, as reflected by similar values for leakageand bursting pressures.

Exposure of the tissue to the NIR laser for 5 and 7 minutes resulted insimilar tissue leaking/bursting pressures; however, tissue charring andshrinkage were observed after irradiation for 7 minutes. Overall, laserirradiation of nanocomposites (GNR 5.4 wt %) for 5 minutes providedoptimal tissue welding, and resulted in tissue leaking and burstingpressure recovery from 3% up to 71% and 45% of the their original intactforms, respectively.

Bacteria leakage. Leakage of bacteria from intestinal tissue wasinvestigated following incision closure using nanocomposite-assistedlaser welding. Based on previous optimization, nanocomposites (˜2 mg)were employed at a fixed GNR concentration of 5.4 wt %, to weld a 5 mmincision located at the center of tubular porcine small intestine (˜10cm in length) using NIR laser irradiation (20 W/cm², 5 min). DH5-α E.coli cells were employed as model bacteria to mimic the inner conditionof the intestine. Note that the bacterial concentration in intestinesections is 10⁵-10⁹ bacteria/gram of intestinal contents[9, 10]; E. colicell cultures with an OD600 of 0.5 is approximately 4*10⁸ bacteria/mL.Leakage of DH5-α cells from inside the intestine to the surroundingfresh LB culture broth was followed as an indication of resistance toinfection.

Rupture of the small intestine resulted in leakage of DH5-α cells intofresh LB broth leading to increase in turbidity of the surroundingmedium as measured using optical density at 600 nm or OD600. No leakagewas observed in case of the intact intestine and the ruptured intestinetreated with the nanocomposite and NIR laser irradiation two hours afterintroducing DH5-α cells (10 mL, OD₆₀₀=0.5) into the tubular smallintestines. In these cases, the fresh LB broth remained clear ornon-turbid. Conversely, the untreated ruptured intestine and rupturedintestine treated with laser alone (without nanocomposite) did notprevent leakage of bacteria; a significant increase in LB broth opticaldensity was observed.

FIGS. 22A and 22B show a quantitative analysis of the bacterial leakage,based on OD₆₀₀. Bacterial leakage was immediately observed uponintroduction of DH5-α cells into the tubular intestines in case ofuntreated ruptured intestine, ruptured intestine treated with the NIRlaser alone (no nanocomposite), and ruptured intestine treated withnanocomposite alone (no laser) conditions (FIG. 22A, unfilled markers).In all these cases, OD₆₀₀ of the fresh LB broth increased from 0 at 0 hto 0.22±0.03 at 4 h, indicating growth of leaked bacterial in the freshLB broth medium. These initial increases in optical density of fresh LBbroth were followed by a gradual decrease in OD₆₀₀ from 0.22±0.03 to0.09±0.03 between 4 and 8 hours, which is likely due to the stationaryand autolytic phases of DH5-α cells[11, 12]. Finally, a steady increasein OD₆₀₀ from 0.09±0.03 up 0.66±0.16 was observed between 8 and 24 hours(FIG. 22B, unfilled markers). This reflects the growth of pre-existingbacteria in the intestinal tissues.

For the condition where ruptured intestine was welded with bothnanocomposite and NIR laser (FIG. 22A, circle filled markers), theoptical density (OD₆₀₀) of the fresh LB broth remained low at 0.04±0.007up to 8 hours after introduction of DH5-αcells into the tubularintestine, indicating no DH5-α leakage. An increase in optical density(up to 0.72±0.25) at 600 nm was observed after 8 h (FIG. 22B, circlefilled markers). Both controls, intact intestine filled with eitherDH5-α cells or fresh LB broth without bacteria (FIG. 22B, square andtriangle filled markers), showed a growth in turbidity similar to thenanocomposite-assisted, laser-welded ruptured intestine samples (FIG.22B, circle filled markers), confirming that bacterial growth between 8and 24 hours is due to pre-existing bacteria in the tissue, and not fromthe leakage of DH5-α cells into fresh LB broth medium. In addition, theoverall growth of bacteria in conditions associated with DH5-α leakage(FIG. 22B, unfilled markers) is less pronounced that those without DH5-αleakage (FIG. 22B, filled markers). This is presumably due to thedepletion of nutrients and the growth competition between DH5-α andintestinal bacteria associated with the tissues. Overall, rupturedintestines that underwent laser tissue welding using nanocomposites canprovide a fluid-tight sealing and prevent bacterial leakage. The leakageprevention was successful and it was observed that the laser-activatednanocomposite continued to provide a liquid-tight sealing for at leastup to one week, which was the duration of these studies (not shown).

Pulsed vs. Continuous Laser. Incised tissue samples were irradiated for1, 3, 5, and 7 min using a continuous wave laser adjusted to 800 nmfollowing application of ELP-GNR matrices (5.4 GNR wt %). Additionally,a tissue sample without an ELP-GNR matrix was irradiated with continuouswave laser adjusted to 800 nm for 7 min. Following irradiation, the allELP-GNR solder was removed and tissue samples were examined. In allcases where the ELP-GNR matrix was present, the wound appeared to haveclosed and there was visible charring around the wound site. Beforeremoval of the ELP-GNR matrices, IR images showed that the temperatureincreased when the samples were irradiated, the greatest temperatureimmediately after irradiation was 107° F. after 5 min of ELP-GNR laserwelding.

Similarly, incised tissue samples were irradiated for 1, 3, 5, and 7 minusing a pulsed laser adjusted to 800 nm following application of ELP-GNRmatrices (5.4 GNR wt %). Following irradiation, it was found that for 5min irradiation, most of the ELP-GNR solder was able to be removed,however some remained welded to the tissue. Charring was visible aroundthe solder-wound site. For the 7 min irradiated sample, it was notpossible to remove any of the solder from the tissue and extent ofcharring was not visible. Before removal of ELP-GNR matrices, IR imagesshowed that immediately after 5 min irradiation, the maximum temperaturereached was temperature ˜148° F. and immediately following 7 minirradiation the temperature was ˜171° F.

Histology. H and E histology was performed on porcine intestine samples.Control samples were prepared where a tissue sample was incised similarto the previous described incision model with the absence of applicationof the nanocomposites, and laser irradiation, a tissue sample was placeddirectly over a flame for 30 seconds, and a tissue sample was “branded”with the heated portion of a metal spatula. Histology images of thecontrol burn samples show that despite excessive heat exposure, theredoes not appear to be major depth of thermal damage. There is a thinlayer of charred tissue at the edge of the tissue and there does appearto be a darkening of the tissue closer to the edges.

Two porcine intestine tissues were cut and placed end-to-end and a5.4%-GNR-C₁₂ELP nanocomposites was placed over the incised portion. Onesample was irradiated with continuous wave laser for 3 minutes and theother was irradiated with pulsed laser for 3 minutes. H&E staining wasperformed on the samples.

Both the continuous wave and the pulsed samples are characterized bythree distinct region. The tissue region is observed at a pinkish color,the nanocomposite region is observed as a purple color, and an adhesionline region, there the patch and the tissue are joined is observed as alighter purple in between the first to regions. Similar to the controlburn samples, in the pulsed samples there seems to be a darkening oftissue nearest to the nanocomposites patch.

C₁₂ELP-GNR-Collagen Nanocomposites. In order to tune the nanocompositesmechanical properties, the inclusion of collagen into the nanocompositessystem was investigated. A 75%-25% C₁₂ELP-Collagen nanocomposite (1.5 mgC₁₂ELP, 0.5 mg Collagen, and 115 μg GNRs) was synthesized.

REFERENCES

-   1. Huang, H C, Yang, Y, Nanda, A, Koria, P, Rege, K. Synergistic    administration of photothermal therapy and chemotherapy to cancer    cells using polypeptide-based degradable plasmonic matrices.    Nanomedicine (Lond) 2011; 6(3): 459-73.-   2. Huang, H C, Koria, P, Parker, S M, Selby, L, Megeed, Z, Rege, K.    Optically responsive gold nanorod-polypeptide assemblies. Langmuir    2008; 24(24): 14139-44.-   3. Karanjia, N D, Corder, A P, Beam, P, Heald, R J. Leakage from    Stapled Low Anastomosis after Total Mesorectal Excision for    Carcinoma of the Rectum. British Journal of Surgery 1994; 81(8):    1224-1226.-   4. Isbister, W H. Anastomotic leak in colorectal surgery: A single    surgeon's experience. Anz Journal of Surgery 2001; 71(9): 516-520.-   5. Park, U. Influence of anastomotic leakage on oncological outcome    in patients with rectal cancer. J Gastrointest Surg 2010; 14(7):    1190-6.-   6. Thomson, G A. An investigation of leakage tracts along stressed    suture lines in phantom tissue. Medical Engineering & Physics 2007;    29(9): 1030-1034.-   7. Zuger, B J, Ott, B, Mainil-Varlet, P, Schaffner, T, Clemence, J    F, Weber, H P, Frenz, M. Laser solder welding of articular    cartilage: Tensile strength and chondrocyte viability. Lasers in    Surgery and Medicine 2001; 28(5): 427-434.-   8. Wolf-de Jonge, I C D, Beek, J F, Balm, R. 25 years of laser    assisted vascular anastomosis (LAVA): What have we learned? European    Journal of Vascular and Endovascular Surgery 2004; 27(5): 466-476.-   9. Spector, D, Rabi, Y, Vasserman, I, Hardy, A, Klausner, J, Rabau,    M, Katzir, A. In Vitro Large Diameter Bowel Anastomosis Using a    Temperature Controlled Laser Tissue Soldering System and Albumin    Stent. Lasers in Surgery and Medicine 2009; 41(7): 504-508.-   10. Asencioarana, F, Garciafons, V, Torresgil, V, Molinaandreu, E,    Vidalmartinez, J, Perersarrio, R, Martinersoriano, F. Effects of a    Low-Power He—Ne-Laser on the Healing of Experimental Colon    Anastomoses—Our Experience. Optical Engineering 1992; 31(7):    1452-1457.-   11. Matteini, P, Rossi, F, Menabuoni, L, Pini, R. Microscopic    characterization of collagen modifications induced by    low-temperature diode-laser welding of corneal tissue. Lasers in    Surgery and Medicine 2007; 39(7): 597-604.-   12. Wadia, Y, Xie, H, Kajitani, M. Liver repair and hemorrhage    control by using laser soldering of liquid albumin in a porcine    model. Lasers in Surgery and Medicine 2000; 27(4): 319-328.-   13. Kirsch, A J, Miller, M I, Hensle, T W, Chang, D T, Shabsigh, R,    Olsson, C A, Connor, J P. Laser-Tissue Soldering in Urinary-Tract    Reconstruction—First Human-Experience. Urology 1995; 46(2): 261-266.-   14. Schober, R, Ulrich, F, Sander, T, Durselen, H, Hessel, S.    Laser-Induced Alteration of Collagen Substructure Allows    Microsurgical Tissue Welding. Science 1986; 232(4756): 1421-1422.-   15. Gobin, A M, O'Neal, D P, Watkins, D M, Halas, N J, Drezek, R A,    West, J L. Near infrared laser-tissue welding using nanoshells as an    exogenous absorber. Lasers in Surgery and Medicine 2005; 37(2):    123-129.-   16. Matteini, P, Ratto, F, Rossi, F, Cicchi, R, Stringari, C,    Kapsokalyvas, D, Pavone, F S, Pini, R. Photothermally-induced    disordered patterns of corneal collagen revealed by SHG imaging.    Optics Express 2009; 17(6): 4868-4878.-   17. Bass, L S, Moazami, N, Pocsidio, J, Oz, MC, Logerfo, P, Treat,    M R. Changes in Type-I Collagen Following Laser-Welding. Lasers in    Surgery and Medicine 1992; 12(5): 500-505.-   18. Murray, L W, Su, L, Kopchok, G E, White, R A. Crosslinking of    Extracellular-Matrix Proteins—a Preliminary-Report on a Possible    Mechanism of Argon-Laser Welding. Lasers in Surgery and Medicine    1989; 9(5): 490-496.-   19. Guillou, P J, Quirke, P, Thorpe, H, Walker, J, Jayne, D G,    Smith, A M H, Heath, R M, Brown, J M. Short-term endpoints of    conventional versus laparoscopic-assisted surgery in patients with    colorectal cancer (MRC CLASICC trial): multicentre, randomised    controlled trial. Lancet 2005; 365(9472): 1718-1726.-   20. Smith, R L, Bohl, J K, McElearney, S T, Friel, C M, Barclay, M    M, Sawyer, R G, Foley, E F. Wound infection after elective    colorectal resection. Annals of Surgery 2004; 239(5): 599-605.-   21. Nelson, H, Sargent, D, Wieand, H S, Fleshman, J, Anvari, M,    Stryker, S J, Beart, R W, Hellinger, M, Flanagan, R, Peters, W, Ota,    D, Hellinger, M. A comparison of laparoscopically assisted and open    colectomy for colon cancer. New England Journal of Medicine 2004;    350(20): 2050-2059.-   22. MacRae, H M, McLeod, R S. Handsewn vs. stapled anastomoses in    colon and rectal surgery—A meta-analysis. Diseases of the Colon &    Rectum 1998; 41(2): 180-189.-   23. Beuran, M, Chiotoroiu, A L, Chilie, A, Morteanu, S, Vartic, M,    Avram, M, Rosu, O, Lica, I. Stapled vs. hand-sewn colorectal    anastomosis in complicated colorectal cancer—a retrospective study.    Chirurgia 2010; 105(5): 645-651.-   24. Park, I J. Influence of Anastomotic Leakage on Oncological    Outcome in Patients with Rectal Cancer. Journal of Gastrointestinal    Surgery; 14(7): 1190-1196.-   25. Anderson, R H. Endoscopic laser surgery handbook (Science and    practice of surgery series, vol. 10). International journal of    cardiology 1988; 20(1): 157.-   26. Cilesiz, I, Springer, T, Thomsen, S, Welch, A J. Controlled    temperature tissue fusion: Argon laser welding of canine intestine    in vitro. Lasers in Surgery and Medicine 1996; 18(4): 325-334.-   27. Chikamatsu, E, Sakurai, T, Nishikimi, N, Yano, T, Nimura, Y.    Comparison of Laser Vascular Welding, Interrupted Sutures, and    Continuous Sutures in Growing Vascular Anastomoses. Lasers in    Surgery and Medicine 1995; 16(1): 34-40.-   28. Gennaro, M, Ascer, E, Mohan, C, Wang, S. A Comparison of Co-2    Laser-Assisted Venous Anastomoses and Conventional Suture    Techniques—Patency, Aneurysm Formation, and Histologic Differences.    Journal of Vascular Surgery 1991; 14(5): 605-613.-   29. Grubbs, P E, Wang, S, Marini, C, Basu, S, Rose, D M, Cunningham,    J N. Enhancement of Co2-Laser Microvascular Anastomoses by Fibrin    Glue. Journal of Surgical Research 1988; 45(1): 112-119.-   30. Capon, A, Iarmarcovai, G, Gonnelli, D, Degardin, N, Magalon, G,    Mordon, S. Scar Prevention Using Laser-Assisted Skin Healing (LASH)    in Plastic Surgery. Aesthetic Plastic Surgery 2010; 34(4): 438-446.-   31. Matteini, P, Ratto, F, Rossi, F, Rossi, G, Esposito, G, Puca, A,    Albanese, A, Maira, G, Pini, R. In vivo carotid artery closure by    laser activation of hyaluronan-embedded gold nanorods. Journal of    Biomedical Optics 2010; 15(4): 0415081-0415086.-   32. Matteini, P, Ratto, F, Rossi, F, Centi, S, Dei, L, Pini, R.    Chitosan Films Doped with Gold Nanorods as Laser-Activatable Hybrid    Bioadhesives. Advanced Materials 2010; 22(38): 4313-4316.-   33. LaJoie, E N, Barofsky, A D, Gregory, K W, Prahl, S A. Patch    welding with a pulsed diode laser and indocyanine green. Lasers in    Medical Science 1997; 12(1): 49-54.-   34. Poppas, D, Sutaria, P, Sosa, R E, Mininberg, D, Schlossberg, S.    Chromophore Enhanced Laser-Welding of Canine Ureters in-Vitro Using    a Human Protein Solder—a Preliminary Step for Laparoscopic Tissue    Welding. Journal of Urology 1993; 150(3): 1052-1055.-   35. Lauto, A, Foster, L J R, Ferris, L, Avolio, A, Zwaneveld, N,    Poole-Warren, L A. Albumin-genipin solder for laser tissue repair.    Lasers in Surgery and Medicine 2004; 35(2): 140-145.-   36. Cilesiz, I, Thomsen, S, Welch, A J, Chan, E K. Controlled    temperature tissue fusion: Ho:YAG laser welding of rat intestine in    vivo .2. Lasers in Surgery and Medicine 1997; 21(3): 278-286.-   37. Cilesiz, I, Thomsen, S, Welch, A J. Controlled temperature    tissue fusion: Argon laser welding of rat intestine in vivo .1.    Lasers in Surgery and Medicine 1997; 21(3): 269-277.-   38. Poppas, D P, Massicotte, J M, Stewart, R B, Roberts, A B, Atala,    A, Retik, A B, Freeman, M R. Human albumin solder supplemented with    TGF-beta(1) accelerates healing following laser welded wound    closure. Lasers in Surgery and Medicine 1996; 19(3): 360-368.-   39. Poppas, D P, Stewart, R B, Massicotte, M, Wolga, A E, Kung, R T    V, Retik, A B, Freeman, M R. Temperature-controlled laser    photocoagulation of soft tissue: In vivo evaluation using a tissue    welding model. Lasers in Surgery and Medicine 1996; 18(4): 335-344.-   40. Cilesiz, I. Controlled temperature phototheramal issue welding.    Journal of Biomedical Optics 1999; 4(3): 327-336.-   41. Klioze, S D, Poppas, D P, Rooke, C T, Choma, T J, Schlossberg,    S M. Development and Initial Application of a Real-Time Thermal    Control-System for Laser-Tissue Welding. Journal of Urology 1994;    152(2): 744-748.-   42. Pasternak, B, Rehn, M, Andersen, L, Agren, M, Heegaard, A-M,    Tengvall, P, Aspenberg, P. Doxycycline-coated sutures improve    mechanical strength of intestinal anastomoses. International Journal    of Colorectal Disease 2008; 23(3): 271-276.-   43. Agren, M, Andersen, T, Andersen, L, Schiodt, C, Surve, V,    Andreassen, T, Risteli, J, Franzen, L, Delaissé, J-M, Heegaard, A-M,    Jorgensen, L. Nonselective matrix metalloproteinase but not tumor    necrosis factor-a inhibition effectively preserves the early    critical colon anastomotic integrity. International Journal of    Colorectal Disease 2011; 26(3): 329-337.-   44. Dallas, P, Sharma, V K, Zboril, R. Silver polymeric    nanocomposites as advanced antimicrobial agents: Classification,    synthetic paths, applications, and perspectives. Advances in Colloid    and Interface Science; 166(1,Äi2): 119-135.-   45. Huang, X H, Jain, P K, El-Sayed, I H, El-Sayed, M A. Plasmonic    photothermal therapy (PPTT) using gold nanoparticles. Lasers in    Medical Science 2008; 23(3): 217-228.-   46. Hu, M, Chen, J Y, Li, Z Y, Au, L, Hartland, G V, Li, X D,    Marquez, M, Xia, Y N. Gold nanostructures: engineering their    plasmonic properties for biomedical applications. Chemical Society    Reviews 2006; 35(11): 1084-1094.-   47. Weissleder, R. A clearer vision for in vivo imaging. Nature    Biotechnology 2001; 19(4): 316-317.-   48. Shao, X, Zheng, W, Huang, Z. Near-infrared autofluorescence    spectroscopy for in vivo identification of hyperplastic and    adenomatous polyps in the colon. Biosensors and Bioelectronics 2011;    30(1): 118-122.-   49. Alencar, H, Funovics, M A, Figueiredo, J, Sawaya, H, Weissleder,    R, Mahmood, U. Colonic Adenocarcinomas: Near-Infrared Microcatheter    Imaging of Smart Probes for Early Detection, AIStudy in Micel.    Radiology 2007; 244(1): 232-238.-   50. Mackay, J A, Chilkoti, A. Temperature sensitive peptides:    Engineering hyperthermia-directed therapeutics. International    Journal of Hyperthermia 2008; 24(6): 483-495.-   51. Nettles, D L, Chilkoti, A, Setton, L A. Applications of    elastin-like polypeptides in tissue engineering. Advanced Drug    Delivery Reviews 2010; 62(15): 1479-1485.-   52. Kim, W, Chaikof, E L. Recombinant elastin-mimetic biomaterials:    Emerging applications in medicine. Advanced Drug Delivery Reviews    2010; 62(15): 1468-1478.-   53. McDaniel, J R, Callahan, D J, Chilkoti, A. Drug delivery to    solid tumors by elastin-like polypeptides. Advanced Drug Delivery    Reviews 2010; 62(15): 1456-1467.-   54. Koria, P, Yagi, H, Kitagawa, Y, Megeed, Z, Nahmias, Y, Sheridan,    R, Yarmush, M L. Self-assembling elastin-like peptides growth factor    chimeric nanoparticles for the treatment of chronic wounds. Proc    Natl Acad Sci USA 2011; 108(3): 1034-9.-   55. McHale, M K, Setton, L A, Chilkoti, A. Synthesis and in vitro    evaluation of enzymatically cross-linked elastin-like polypeptide    gels for cartilaginous tissue repair. Tissue Engineering 2005;    11(11-12): 1768-1779.-   56. Pasternak, B, Matthiessen, P, Jansson, K, Andersson, M,    Aspenberg, P. Elevated intraperitoneal matrix metalloproteinases-8    and -9 in patients who develop anastomotic leakage after rectal    cancer surgery: a pilot study. Colorectal Disease 2010; 12(7Online):    e93-e98.-   57. Kim, J C, Lee, Y K, Lim, B S, Rhee, S H, Yang, H C. Comparison    of tensile and knot security properties of surgical sutures. Journal    of Materials Science-Materials in Medicine 2007; 18(12): 2363-2369.-   58. Watters, D A K, Smith, A N, Eastwood, M A, Anderson, K C, Elton,    R A, Mugerwa, J W. Mechanical-Properties of the Colon—Comparison of    the Features of the African and European Colon Invitro. Gut 1985;    26(4): 384-392.-   59. Teng, W, Cappello, J, Wu, X. Recombinant Silk-Elastinlike    Protein Polymer Displays Elasticity Comparable to Elastin.    Biomacromolecules 2009; 10(11): 3028-3036.-   60. Rnjak-Kovacina, J, Wise, SG, Li, Z, Maitz, P K M, Young, C J,    Wang, Y, Weiss, A S. Tailoring the porosity and pore size of    electrospun synthetic human elastin scaffolds for dermal tissue    engineering. Biomaterials 2011; 32(28): 6729-6736.-   61. McKenna, K A, Hinds, M T, Sarao, R C, Wu, P-C, Maslen, C L,    Glanville, R W, Babcock, D, Gregory, K W. Mechanical property    characterization of electrospun recombinant human tropoelastin for    vascular graft biomaterials. Acta Biomaterialia 2012; 8(1): 225-233.-   62. Thomsen, S, Morris, J R, Neblett, C R, Mueller, J. Tissue    Welding Using a Low-Energy Microsurgical Co2-Laser. Medical    Instrumentation 1987; 21(4): 231-237.-   63. Chen, C C, Lin, Y P, Wang, C W, Tzeng, H C, Wu, C H, Chen, Y C,    Chen, C P, Chen, L C, Wu, Y C. DNA-gold nanorod conjugates for    remote control of localized gene expression by near infrared    irradiation. Journal of the American Chemical Society 2006; 128(11):    3709-3715.-   64. Ratto, F, Matteini, P, Cini, A, Centi, S, Rossi, F, Fusi, F,    Pini, R. CW laser-induced photothermal conversion and shape    transformation of gold nanodogbones in hydrated chitosan films.    Journal of Nanoparticle Research 2011; 13(9): 4337-4348.-   65. Garcia, P, Mines, M J, Bower, K S, Hill, J, Menon, J, Tremblay,    E, Smith, B. Robotic Laser Tissue Welding of Sclera Using Chitosan    Films. Lasers in Surgery and Medicine 2009; 41(1): 60-67.-   66. Huang, H C, Rege, K, Heys, J J. Spatiotemporal temperature    distribution and cancer cell death in response to extracellular    hyperthermia induced by gold nanorods. ACS Nano 2010; 4(5):    2892-900.-   67. Galya, T, Sedlarik, V, Kuritka, I, Novotny, R, Sedlarikova, J,    Saha, P. Antibacterial Poly(vinyl Alcohol) Film Containing Silver    Nanoparticles: Preparation and Characterization. Journal of Applied    Polymer Science 2008; 110(5): 3178-3185.-   68. Wei, D W, Sun, W Y, Qian, W P, Ye, Y Z, Ma, X Y. The synthesis    of chitosan-based silver nanoparticles and their antibacterial    activity. Carbohydrate Research 2009; 344(17): 2375-2382.-   69. Vimala, K, Mohan, Y M, Sivudu, K S, Varaprasad, K, Ravindra, S,    Reddy, N N, Padma, Y, Sreedhar, B, MohanaRaju, K. Fabrication of    porous chitosan films impregnated with silver nanoparticles: A    facile approach for superior antibacterial application. Colloids and    Surfaces B-Biointerfaces 2010; 76(1): 248-258.-   70. Rai, M, Yadav, A, Gade, A. Silver nanoparticles as a new    generation of antimicrobials. Biotechnology Advances 2009; 27(1):    76-83.-   71. Lippert, E, Klebl, F, Schweller, F, Ott, C, Gelbmann, C,    Scholmerich, J, Endlicher, E, Kullmann, F. Fibrin glue in the    endoscopic treatment of fistulae and anastomotic leakages of the    gastrointestinal tract. International Journal of Colorectal Disease    2011; 26(3): 303-311.-   72. Skardal, A, Zhang, J, McCoard, L, Oottamasathien, S, Prestwich,    G D. Dynamically Crosslinked Gold Nanoparticle—Hyaluronan Hydrogels.    Advanced Materials 2010; 22(42): 4736-4740.-   73. Raub, C B, Putnam, A J, Tromberg, B J, George, S C. Predicting    bulk mechanical properties of cellularized collagen gels using    multiphoton microscopy. Acta Biomaterialia 2010; 6(12): 4657-4665.-   74. Moyer, M P, Manzano, L A, Merriman, R L, Stauffer, J S, Tanzer,    L R. NCM460, a normal human colon mucosal epithelial cell line. In    Vitro Cellular & Developmental Biology-Animal 1996; 32(6): 315-317.-   75. Huang, H C, Barua, S, Kay, D B, Rege, K. Simultaneous    Enhancement of Photothermal Stability and Gene Delivery Efficacy of    Gold Nanorods Using Polyelectrolytes. Acs Nano 2009; 3(10):    2941-2952.-   76. Barua, S, Joshi, A, Banerjee, A, Matthews, D, Sharfstein, S T,    Cramer, S M, Kane, R S, Rege, K. Parallel Synthesis and Screening of    Polymers for Nonviral Gene Delivery. Molecular Pharmaceutics 2009;    6(1): 86-97.-   77. Liang, C C, Park, A Y, Guan, J L. In vitro scratch assay: a    convenient and inexpensive method for analysis of cell migration in    vitro. Nature Protocols 2007; 2(2): 329-333.-   78. Jung, H C, Eckmann, L, Yang, S K, Panja, A, Fierer, J,    Morzyckawroblewska, E, Kagnoff, M F. A Distinct Array of    Proinflammatory Cytokines Is Expressed in Human Colon    Epithelial-Cells in Response to Bacterial Invasion. Journal of    Clinical Investigation 1995; 95(1): 55-65.-   79. Eckmann, L, Jung, H C, Schurermaly, C, Panja, A,    Morzyckawroblewska, E, Kagnoff, M F. Differential Cytokine    Expression by Human Intestinal Epithelial-Cell Lines—Regulated    Expression of Interleukin-8. Gastroenterology 1993; 105(6):    1689-1697.-   80. Werner, S, Grose, R. Regulation of wound healing by growth    factors and cytokines. Physiological Reviews 2003; 83(3): 835-870.-   81. Wu, Y, MacKay, J A, McDaniel, J R, Chilkoti, A, Clark, R L.    Fabrication of Elastin-Like polypeptide Nanoparticles for Drug    Delivery by Electrospraying. Biomacromolecules 2009; 10(1): 19-24.-   82. Huang, L, McMillan, R A, Apkarian, R P, Pourdeyhimi, B,    Conticello, V P, Chaikof, E L. Generation of synthetic    elastin-mimetic small diameter fibers and fiber networks.    Macromolecules 2000; 33(8): 2989-2997.-   83. Qiu, W G, Teng, WB, Cappello, J Y, Wu, X. Wet-Spinning of    Recombinant Silk-Elastin-Like Protein Polymer Fibers with High    Tensile Strength and High Deformability. Biomacromolecules 2009;    10(3): 602-608.-   84. Anumolu, R, Gustafson, J A, Magda, J J, Cappello, J, Ghandehari,    H, Pease, L F. Fabrication of Highly Uniform Nanoparticles from    Recombinant Silk-Elastin-like Protein Polymers for Therapeutic Agent    Delivery. ACS Nano 2011; 5(7): 5374-5382.-   85. MacEwan, S R, Chilkoti, A. Elastin-like polypeptides: Biomedical    applications of tunable biopolymers. Peptide Science 2010; 94(1):    60-77.-   86. Rubert Perez, C M, Panitch, A, Chmielewski, J. A Collagen    Peptide-Based Physical Hydrogel for Cell Encapsulation.    Macromolecular Bioscience 2011; 11(10): 1426-1431.

REFERENCES FOR EXAMPLE 3

-   ¹ Overgaard J: The current and potential role of hyperthermia in    radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 16(3), 535-549    (1989).-   ² Huang X H, Jain P K, El-Sayed I H, El-Sayed M A: Determination of    the minimum temperature required for selective photothermal    destruction of cancer cells with the use of immunotargeted gold    nanoparticles. Photochem. Photobiol. 82(2), 412-417 (2006).-   ³ He X M, Wolkers W F, Crowe J H, Swanlund D J, Bischof J C: In situ    thermal denaturation of proteins in dunning at-1 prostate cancer    cells: implication for hyperthermic cell injury. Ann. Biomed. Engin.    32(10), 1384-1398 (2004).-   ⁴ Lepock J R: Cellular effects of hyperthermia: relevance to the    minimum dose for thermal damage. Int. J. Hypertherm. 19(3), 252-266    (2003).-   ⁵ Seki T, Wakabayashi M, Nakagawa T et al.: Percutaneous microwave    coagulation therapy for solitary metastatic liver tumors from    colorectal cancer: a pilot clinical study. Am. J. Gastroenterol.    94(2), 322-327 (1999).-   6 Gazelle G S, Goldberg S N, Solbiati L, Livraghi T: Tumor ablation    with radiofrequency energy. Radiology 217(3), 633-646 (2000).-   7 Hilger I, Andra W, Bahring R, Daum A, Hergt R, Kaiser W A:    Evaluation of temperature increase with different amounts of    magnetite in liver tissue samples. Investig. Radiol. 32(11), 705-712    (1997).-   ⁸ Jolesz F A, Hynynen K: Magnetic resonance image-guided focused    ultrasound surgery. Cancer J. 8, S100-S112 (2002).-   ⁹ Dickerson E B, Dreaden E C, Huang X et al.: Gold nanorod assisted    near-infrared plasmonic photothermal therapy (PPTT) of squamous cell    carcinoma in mice. Cancer Lett. 269(1), 57-66 (2008).-   ¹⁰ Gobin A M, Lee M R, Halas N J, James W D, Drezek R A, West J L:    Near-infrared resonant nanoshells for combined optical imaging and    photothermal cancer therapy. Nano Lett. 7(7), 1929-1934 (2007).-   ¹¹ Skrabalak S E, Chen J, Sun Y et al.: Gold nanocages: synthesis,    properties, and applications. Accounts Chem. Res. 41(12), 1587-1595    (2008).-   ¹² Huang X, Jain P K, El-Sayed I H, El-Sayed M A: Plasmonic    photothermal therapy (PPTT) using gold nanoparticles. Lasers Med.    Sci. 23(3), 217-228 (2008).-   ¹³ Huang X, El-Sayed I H, Qian W, El-Sayed M A: Cancer cell imaging    and photothermal therapy in the near-infrared region by using gold    nanorods. J. Am. Chem. Soc. 128(6), 2115-2120 (2006).-   ¹⁴ Von Maltzahn G, Park J H, Agrawal A et al.: Computationally    guided photothermal tumor therapy using long-circulating gold    nanorod antennas. Cancer Res. 69(9), 3892-3900 (2009).-   ¹⁵ Ma L L, Feldman M D, Tam J M et al.: Small multifunctional    nanoclusters (nanoroses) for targeted cellular imaging and therapy.    ACS Nano 3(9), 2686-2696 (2009).-   ¹⁶ Tong L, Wei Q, Wei A, Cheng J-X: Gold nanorods as contrast agents    for biological imaging: optical properties, surface conjugation and    photothermal effects. Photochem. Photobiol. 85(1), 21-32 (2009).-   ¹⁷ Lepock J R: Cellular effects of hyperthermia: Relevance to the    minimum dose for thermal damage. Int. J. Hypertherm. 19(3), 252-266    (2003).-   ¹⁸ Gibbons N B, Watson R W, Coffey R N, Brady H P, Fitzpatrick J M:    Heat-shock proteins inhibit induction of prostate cancer cell    apoptosis. Prostate 45(1), 58-65 (2000).-   ¹⁹ Rylander M N, Feng Y, Bass J, Diller K R: Thermally induced    injury and heat-shock protein expression in cells and tissues. Ann.    NY Acad. Sci. 1066, 222-242 (2005).-   ²⁰ Heath E I, Hillman D W, Vaishampayan U et al.: A Phase II trial    of 17-allylamino-17-demethoxygeldanamycin in patients with    hormone-refractory metastatic prostate cancer. Clin. Cancer Res.    14(23), 7940-7946 (2008).-   ²¹ Solit D B, Osman I, Polsky D et al.: Phase II trial of    17-allylamino-17-demethoxygeldanamycin in patients with metastatic    melanoma. Clin. Cancer Res. 14(24), 8302-8307 (2008).-   ²² Nikoobakht B, El-Sayed M A: Preparation and growth mechanism of    gold nanorods (NRs) using seed-mediated growth method. Chem. Mater.    15(10), 1957-1962 (2003).-   ²³ Huang H-C, Koria P, Parker S M, Selby L, Megeed Z, Rege K:    Optically responsive gold nanorod—polypeptide assemblies. Langmuir    24(24), 14139-14144 (2008).-   ²⁴ Rege K, Patel S J, Megeed Z, Yarmush M L: Amphipathic    peptide-based fusion peptides and immunoconjugates for the targeted    ablation of prostate cancer cells. Cancer Res. 67(13), 6368-6375    (2007).-   ²⁵ Barua S, Joshi A, Banerjee A et al.: Parallel synthesis and    screening of polymers for nonviral gene delivery. Mol. Pharm. 6(1),    86-97 (2009).-   ²⁶ Gong M C, Latouche J B, Krause A, Heston W D, Bander N H,    Sadelain M: Cancer patient T cells genetically targeted to    prostate-specific membrane antigen specifically lyse prostate cancer    cells and release cytokines in response to prostate-specific    membrane antigen. Neoplasia 1(2), 123-127 (1999).-   ²⁷ Urry D W: Physical chemistry of biological free energy    transduction as demonstrated by elastic protein-based polymers. J.    Phys. Chem. B 101(51), 11007-11028 (1997).-   ²⁸ Meyer D E, Chilkoti A: Genetically encoded synthesis of    protein-based polymers with precisely specified molecular weight and    sequence by recursive directional ligation: examples from the    elastin-like polypeptide system. Biomacromolecules 3(2), 357-367    (2002).-   ²⁹ Janorkar A V, Rajagopalan P, Yarmush M L, Megeed Z: The use of    elastin-like polypeptide-polyelectrolyte complexes to control    hepatocyte morphology and function in vitro. Biomaterials 29(6),    625-632 (2008).-   ³⁰ Huang H-C, Barua S, Kay D B, Rege K: Simultaneous enhancement of    photothermal stability and gene delivery efficacy of gold nanorods    using polyelectrolytes. ACS Nano 3(10), 2941-2952 (2009).-   ³¹ Huang Hc, Rege K, Heys J J: Spatiotemporal temperature    distribution and cancer cell death in response to extracellular    hyperthermia induced by gold nanorods. ACS Nano 4(5), 2892-2900    (2009).-   ³² Huang X, El-Sayed I H, Qian W, El-Sayed M A: Cancer cell imaging    and photothermal therapy in the near-infrared region by using gold    nanorods. J. Am. Chem. Soc. 128(6), 2115-2120 (2006).-   33. Lowery A R, Gobin A M, Day E S, Halas N J, West J L:    Immunonanoshells for targeted photothermal ablation of tumor cells.    Int. J. Nanomed. 1(2), 149-154 (2006).

REFERENCES FOR EXAMPLE 5

-   1. Park, H., et al., Effect of Swelling Ratio of Injectable Hydrogel    Composites on Chondrogenic Differentiation of Encapsulated Rabbit    Marrow Mesenchymal Stem Cells In Vitro. Biomacromolecules, 2009.    10(3): p. 541-546.-   2. Cilesiz, I., et al., Controlled temperature tissue fusion: Argon    laser welding of canine intestine in vitro. Lasers in Surgery and    Medicine, 1996. 18(4): p. 325-334.-   3. Poppas, D. P., et al., Temperature-controlled laser    photocoagulation of soft tissue: In vivo evaluation using a tissue    welding model. Lasers in Surgery and Medicine, 1996. 18(4): p.    335-344.-   4. Wise, L., W. McAlister, and T. Stein, Studies on the healing of    anastomoses of small and large intestines. Surg Gynecol    Obstet, 1975. 141(190).-   5. Pasternak, B., et al., Doxycycline-coated sutures improve    mechanical strength of intestinal anastomoses. International Journal    of Colorectal Disease, 2008. 23(3): p. 271-276.-   6. Lipska, M. A., et al., Anastomotic leakage after lower    gastrointestinal anastomosis: Men are at a higher risk. Anz Journal    of Surgery, 2006. 76(7): p. 579-585.-   7. Post, S., et al., Risks of Intestinal Anastomoses in    Crohns-Disease. Annals of Surgery, 1991. 213(1): p. 37-42.-   8. Pickleman, J., et al., The failed gastrointestinal anastomosis:    An inevitable catastrophe? Journal of the American College of    Surgeons, 1999. 188(5): p. 473-482.-   9. Hooper, L. V., et al., Molecular analysis of commensal    host-microbial relations hips in the intestine. Science, 2001.    291(5505): p. 881-884.-   10. Husebye, E., et al., Influence of microbial species on small    intestinal myoelectric activity and transit in germ-free rats.    American Journal of Physiology-Gastrointestinal and Liver    Physiology, 2001. 280(3): p. G368-G380.-   11. Leduc, M., R. Kasra, and J. Vanheijenoort, Induction and Control    of the Autolytic System of Escherichia-Coli. Journal of    Bacteriology, 1982. 152(1): p. 26-34.-   12. Leduc, M. and J. Vanheijenoort, Autolysis of Escherichia-Coli.    Journal of Bacteriology, 1980. 142(1): p. 52-59.

What is claimed is:
 1. A bioadhesive composition comprising: aphotothermally responsive composition comprising a network ofelastin-like polypeptide (ELP) and a light absorbing chromophore,wherein the light absorbing chromophore comprises one or more metallicnanoparticles physically entrapped within the network of ELP.
 2. Thebioadhesive composition of claim 1, wherein the one or more metallicnanoparticles comprise silver nanoparticles, gold nanorods, or goldnanoparticles, or mixtures thereof.
 3. The bioadhesive composition ofclaim 1, further comprising an active agent, cells, or a combinationthereof physically entrapped within the network of ELP.
 4. Thebioadhesive composition of claim 3, wherein the active agent comprisesan antibacterial agent.
 5. The bioadhesive composition of claim 3,wherein the active agent comprises an MMP inhibitor, a soluble factor, acytokine, or a growth factor.
 6. The bioadhesive composition of claim 5,wherein the soluble factor comprises FGF (fibroblast growth factor),TGF-beta, EGF, or a combination thereof.
 7. A bioadhesive compositioncomprising: a photothermally responsive composition comprising a networkof elastin-like polypeptide (ELP) and a light absorbing chromophore,wherein the light absorbing chromophore comprises one or more metallicnanoparticles physically entrapped within the network of ELP, andwherein the light absorbing chromophore is configured to convert lightto heat upon the application of a directed light beam to thephotothermally responsive composition, to a surrounding tissue, or toboth, thereby crosslinking the network of ELP in response to the heat.8. The bioadhesive composition of claim 7, wherein one or more metallicnanoparticles comprise silver nanoparticles, gold nanorods, or goldnanoparticles, or mixtures thereof.
 9. The bioadhesive composition ofclaim 7, wherein the directed light beam is a laser.
 10. The bioadhesivecomposition of claim 7, wherein the light is near infrared.
 11. Thebioadhesive composition of claim 7, wherein the directed light beamgenerates a bulk temperature of at least a portion of the photothermallyresponsive bioadhesive composition above 60° C.
 12. The bioadhesivecomposition of claim 7, wherein the directed light beam generates a bulktemperature of at least a portion of the photothermally responsivebioadhesive composition above 80° C.
 13. The bioadhesive composition ofclaim 7, further comprising an active agent, cells, or a combinationthereof physically entrapped within the fibrous network of ELP, whereinthe photothermally responsive composition is configured to release theactive agent from the fibrous network of ELP upon the application of thedirected light beam.
 14. A suture comprising: at least two fibers,wherein each fiber comprises the bioadhesive composition of claim
 1. 15.The suture of claim 14, wherein the one or more metallic nanoparticlescomprise silver nanoparticles, gold nanorods, or gold nanoparticles, ormixtures thereof.
 16. The suture of claim 14, wherein the directed lightbeam is a laser.
 17. The suture of claim 14, wherein the light is nearinfrared.
 18. The suture of claim 14, wherein the directed light beamgenerates a bulk temperature of at least a portion of the photothermallyresponsive bioadhesive composition above 60° C.
 19. The suture of claim14, wherein the directed light beam generates a bulk temperature of atleast a portion of the photothermally responsive bioadhesive compositionabove 80° C.
 20. The suture of claim 14, wherein the light absorbingchromophore in the at least two fibers has a weight percentage between0.5% and 8%.